Metadherin polypeptides, encoding nucleic acids and methods of use

ABSTRACT

Metadherin, a protein that controls metastasis, and variants of metadherin are described. DNA sequences encoding the same and methods of production are described. Therapies involving the application of metadherin, binding agents that bind to metadherin, such as antibodies, and expression modulating agents, such as siRNA, are described. The use of metadherin or metadherin variants for delivering desired substances to particular lung tissue is described. A method of diagnosing metastatic cells based on the presence of metadherin is described.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/519,675, filed Nov. 13, 2003, hereby incorporated by reference inits entirety.

GOVERNMENTAL INTEREST

This work was supported by grants CA 82713, CA 30199, and CA09579 fromthe National Cancer Institute of the National Institutes of Health, andDAMD17-02-1-0315 from the Department of Defense. The government may havecertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology and molecularmedicine and more specifically to the regulation of tumor metastasis.

BACKGROUND

Tumor metastasis is a complex, multi-step process in which cancer cellsdetach from the original tumor mass and establish metastatic foci atorgan-specific sites (Fidler, I. J., Surg Oncol Clin N Am 10: 257-269,vii-viiii. (2001)). The location of the metastatic site depends on theparticular type of cancer and stage of disease. For example, breastcancer typically spreads first to the lungs and liver (Kamby, C. et al.,Cancer 59: 1524-1529 (1987); Rutgers, E. J. et al., Br J Surg 76:187-190 (1989); Tomin, R. and Donegan, W. L., J Clin Oncol 5: 62-67(1987)). Later in the disease, breast cancer spreads to the centralnervous system and bone (Amer, M. H., J Surg Oncol 19: 101-105 (1982);Boogerd, W., Radiother Oncol 40, 5-22 (1996)). The metastatic phase ofthe disease is devastating, given that conventional treatments areusually ineffective and patients typically survive only a few yearsafter diagnosis (Harris, J. et al., Cancer of the breast. In Cancer,principles and practice of oncology (Philadelphia, Lippincott Co.), pp.1602-1616 (1982)).

Several factors affect the location and growth of metastases. Dependingon the blood-flow pattern from the primary tumor, certain tumor cellsare carried preferentially to particular organs (Weiss, L., Clin ExpMetastasis 10: 191-199 (1992)). While in circulation, some tumor cellsselectively recognize particular endothelial cell surface molecules thatmediate cell adhesion to specific organs (Abdel-Ghany, M. et al., J BiolChem 276: 25438-25446 (2001); Cheng, H. C. et al., J Biol Chem 273:24207-24215 (1998); Johnson, R. C. et al., J Cell Biol 121: 1423-1432(1993)). The arrest of tumor cells at the metastatic site, throughmechanical trapping in small capillaries or through adhesiveinteractions with the endothelium, is a necessary step for tumors tobecome established at a secondary site (Chambers, A. F. et al., Nat RevCancer 2: 563-572 (2002); Orr, F. W. and Wang, H. H., Surg Oncol Clin NAm 10: 357-381, ix-x (2001)). Once the tumor cells have seeded thetarget organ, the local microenvironment influences whether or not aparticular cancer cell will proliferate (Fidler, I. J., Surg Oncol ClinN Am 10: 257-269, vii-viiii (2001); Radinsky, R., Cancer Metastasis Rev14: 323-338 (1995)). Unfortunately, many of the factors that contributeto organ-specific metastasis have yet to be elucidated.

Of the many possible factors, one such possible factor may be theprotein or proteins that allow homing of a protein or a cell to aparticular tissue by binding to a molecule in the tissue. Somelung-specific homing peptides have been isolated by in vivo phagedisplay (Rajotte, D. and Ruoslahti, E., J Biol Chem 274: 11593-11598(1999)) and antibodies that specifically bind to lung vasculature havebeen prepared (McIntosh, D. P., et al., Proc Natl Acad Sci USA 99:1996-2001 (2002)). Tissue-specific expression of vascular markers is notlimited to lung vasculature; recent data suggest that each tissue puts aspecific signature on its vasculature (Ruoslahti, E., Nat Rev Cancer 2:83-90 (2002)). Thus, binding of tumor cells to tissue-specific vascularmarkers may play a role in selective tumor metastasis to other tissuesas well.

There are examples of adhesive interactions that are required in orderfor lung metastases to form. Dipeptidyl dipeptidase IV on lungendothelial cells was found to be an adhesion receptor for fibronectinon metastasizing breast and prostate carcinoma cells in a mouse model(Cheng, H. C. et al., J Biol Chem 273: 24207-24215 (1998); Johnson, R.C., et al., J Cell Biol 121: 1423-1432 (1993)). In another mouse model,Ca²⁺-sensitive chloride channel, hCLCA2, on lung endothelial cells wasreported to be a ligand for β4 integrins on metastasizing breast cancercells (Abdel-Ghany, M., et al., J Biol Chem 276: 25438-25446 (2001);Elble, R. C., et al., J Biol Chem 272: 27853-27861 (1997)). Mostrecently, the secreted chemokine, CXCL12, which is highly expressed inthe lung, liver, and lymph nodes, was shown to bind to CXCR4 receptorson the surface of metastasizing breast cancer cells (Muller et al.,2001). Moreover, interfering with only one of these interactions wassufficient to inhibit metastasis (Abdel-Ghany, M., et al., J Biol Chem276: 25438-25446 (2001); Cheng, H. C. et al., J Biol Chem 273:24207-24215 (1998); Muller, A., et al., Nature 410: 50-56 (2001)).Although there is no evidence available on the significance of theseinteractions in breast cancer, it seems that multiple interactions ofcell adhesion molecules and growth factor receptors may be required forthe attachment and growth of circulating tumor cells. Similar mechanismsbased on unique vascular addresses may play a role in organ-specificmetastasis to other organs.

Others have identified numerous genes, whose expression are increased inmetastatic breast cancer. One such gene, described as GenBank™ entryAK000745 was up-regulated in metastatic breast cancers, along with manyother genes (Van't Veer, et al., Nature 415: 530-536 (2002). However,the possibility of a causal role of this gene or its protein product(herein named metadherin) in breast cancer metastasis has not yet beenestablished.

SUMMARY OF INVENTION

Aspects of the invention relate to variants of metadherin and thenucleic acids and polypeptides that encode metadherin. Thesepolypeptides and nucleic acids are useful for modulating and diagnosingtumor metastasis. Metadherin polypeptides and nucleic acids can beadvantageously used to diagnose or treat cancer, in particular breastcancer and lung cancer.

Furthermore, metadherin polypeptides and nucleic acids encodingmetadherin can be useful to screen for agents that can alter metadherinactivity or expression. In addition, these nucleic acids and proteinscan be used to discover useful general agents and binding agents, suchas small molecule drugs, peptides, antibodies, anti-sense nucleic acids,or small interfering RNA, each of which can affect tumor metastasis.

Some embodiments of the invention may also include vectors containingmetadherin nucleic acids, host cells containing such vectors, metadherinanti-sense nucleic acids and related compositions.

Other embodiments of the invention include a molecule which is able tolocalize desired substances to the lung tissue. In one embodiment, thelocalizing molecule is a fragment of metadherin. In another embodiment,the molecule includes amino acids 378-440 of metadherin.

Yet other embodiments are oligonucleotides that hybridize to, oramplify, a metadherin nucleic acid.

Other embodiments are anti-metadherin specific antibodies and the use ofthese antibodies to treat metastatic cancer. Further provided are kitscontaining metadherin nucleic acids or metadherin specific antibodies,such kits and reagents can be used to diagnose cancer, monitor responseto therapy, or predict the prognosis of a cancer patient.

Also provided are methods of modulating tumor metastasis usingmetadherin polypeptides, encoding nucleic acids, compounds, or agentsthat modulate the activity or expression of metadherin polypeptides. Themethods for modulating tumor metastasis can be used to treat diseasessuch as cancer.

Other embodiments provide methods of therapy involving administeringantisense nucleotides directed to the metadherin sequence. Furtherprovided are methods of treating a patient involving administering, to apatient, antisense nucleotides that are directed to the metadherinsequence.

Other embodiments provide methods of reducing metadherin mediatedlocalization by administering an antibody that is directed tometadherin.

Other embodiments provide methods of reducing the expression ofmetadherin by administering siRNA directed to the metadherin sequence toa sample. Further provided is a method for helping a patient with cancerby administering an effective amount of siRNA to the patient in order toreduce the level of expression of metadherin on the metastatic cells.

Other embodiments provide methods for imaging a cancer in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of the lung-binding domain and structuralinformation of metadherin protein.

FIG. 1A shows an amino acid sequence of metadherin's lung-homing domain.This domain corresponds to residues 378-440 of the full-length mousemetadherin protein.

FIG. 1B shows a hydrophobicity analysis of metadherin, using a windowsize of 9 amino acids.

FIG. 1C shows a layout of the full-length metadherin protein.

FIG. 2 shows an alignment performed on BLAST of human metadherin andseveral metadherin homologs.

FIG. 3 is a graph showing metadherin phage titers recovered from lung,pancreas, breast, skin, kidney, brain, and liver after injection intothe tail-vein of Balb/c mice and circulation for 5 minutes.

FIG. 4 shows that the lung-homing domain of metadherin is extracellular.

FIG. 4A is a graph that shows HEK293T cells expressing full-lengthmyc-tagged metadherin, myc-vimentin, or myc-pCMV that were analyzed byflow cytometry.

FIG. 4B is a graph that shows Rabbit IgG, anti-Bcl2 polyclonal Ab(Bcl2), anti-integrin α5β3 polyclonal antibody (Integrin α5β1), oranti-metadherin₍₃₇₈₋₄₄₀₎(metadherin) as applied to non-permeabilized 4T1tumor cells and detected with a FITC-labeled secondary Ab.

FIG. 5 is an image of a gel that demonstrates metadherin expression in4T1 tumor cells.

FIG. 6 is a graph that shows the number of DsRed2-positive cells perviewing field in the lung sections. (n=75; One-tailed Student's t-test;*P<0.001).

FIG. 7 shows that anti-metadherin antibodies and siRNA reactive tometadherin mRNA inhibit lung metastasis.

FIG. 7A is an image of a gel that shows an anti-myc immunoblot ofHEK293T cell extracts expressing myc-tagged metadherin and siRNAreactive to GAPDH, scrambled mRNA, or metadherin.

FIG. 7B is an image of a gel that shows immunoblot quantitation ofβ-actin and transferrin receptor protein levels in 4T1 cells expressingsiRNA reactive to metadherin or scrambled mRNA.

FIG. 7C is an image of a gel that shows immunoblot quantitation ofmetadherin protein levels in 4T1 cells expressing siRNA reactive tometadherin or scrambled mRNA.

FIG. 7D is a bar graph that shows real time RT-PCR quantitation ofmetadherin mRNA in 4T1 cells expressing siRNA reactive to metadherin orscrambled mRNA.

FIG. 7E is a graph displaying that siRNA reactive to metadherintranscripts inhibits 4T1 cell experimental lung metastasis.

FIG. 7F is a graph that shows the number of lung metastases from miceinjected with 4T1 cells that were treated with anti-metadherin₍₃₇₈₋₄₄₀₎,rabbit IgG, or PBS.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein, metadherin and functional fragments thereof werecharacterized and discovered to be cell-surface proteins thatparticularly recognize lung vasculature and are involved in themetastasis of tumors. One functional fragment is shown in SEQ ID NO: 3and corresponds to amino acids 378-440 of murine metadherin (SEQ ID NO:1). As disclosed herein, metadherin was found to be over-expressed inmetastatic breast tumors and bound to the lung vasculature through aC-terminal segment in the extracellular domain. Accordingly, methods andcompositions are provided for inhibiting tumor metastasis by inhibitingthe expression or function of metadherin. Methods and compositions arealso provided for inhibiting the binding of metadherin to its receptorin the lung vasculature.

Some embodiments of the invention include isolated murine metadherinprotein (SEQ ID NO: 1) or isolated DNA (SEQ ID NO: 2) encoding themetadherin protein, as well as the human homologs (SEQ ID NO: 13 and SEQID NO: 16). In addition, embodiments of the invention include isolatedpolypeptides and functional variants thereof, the nucleic acid moleculesencoding such polypeptides and variants, and related compositions andmethods. Other embodiments include methods and compositions forinhibiting the activity or expression of metadherin. Compositions formodulating the activity or expression of metadherin can include, but arenot limited to, metadherin, variants thereof, antibodies, siRNA,antisense molecules, peptides, proteins, or small molecules.

Methods are provided herein to identify compounds that modulate, eitherpositively or negatively, the activity or expression of metadherin.Embodiments of the invention also include oligonucleotides that can beused to hybridize to or amplify a metadherin nucleic acid. Embodimentsof the invention also include kits containing metadherin nucleic acidsor metadherin specific antibodies. Such kits and reagents can be used todiagnose cancer, monitor response to therapy, or predict the prognosisof a cancer patient.

Another embodiment of the invention is a method for inhibiting tumormetastasis. The method includes selecting a patient who has a risk oftumor metastasis and administering an effective amount of a compoundthat inhibits the binding of metadherin or a variant or fragmentthereof, to the metadherin receptor. The selection of a patient may bedone in many alternative ways. For example, the type of cancer that apatient has may indicate the likelihood of the cancer's spread. In oneembodiment, any patient with cancer, or with a substantial risk ofgetting cancer, may be the subject of this treatment. In anotherembodiment, the identification of the patient is achieved by using themethod of the current embodiments, such as detecting the presence ofexcess metadherin, as described more fully below. In one embodiment, thecompound comprises a binding fragment of metadherin, such as the murinelung homing domain (SEQ ID NO: 3) or the corresponding domain (SEQ IDNO: 17) from amino acids 378-440 of the human metadherin protein (SEQ IDNO: 13). In an alternative embodiment, the compound may be a monoclonalantibody that preferentially binds metadherin. In an alternativeembodiment, the compound may be a peptide, where the peptide bindseither to metadherin or to a receptor of metadherin. In one embodiment,the compound may be a variant of metadherin. In one embodiment, thecompound may be a peptidomimetic, where the peptidomimetic binds eitherto metadherin or to a receptor of metadherin. In an alternativeembodiment, the compound may be an antisense or siRNA molecule thatinhibits expression of metadherin. In an alternative embodiment, thecompound may be a small molecule drug.

The compounds can be delivered to locations depending upon thecompounds' properties, as will be appreciated by one of skill in theart. For example, antibodies, peptides to metadherin, antisense mRNA,siRNA and receptors can be delivered to the location of the presentcancer. Likewise, peptides that bind to the receptor or metadherin,antibodies, receptors or fragments thereof, and metadherin or variantsthereof, may be delivered to the location to which the cancer mayspread. In one embodiment, the cancer is breast cancer. In oneembodiment, the receptor for metadherin is located on the lungvasculature.

Methods are described below for the treatment of a patient sufferingfrom breast cancer, or at risk of breast cancer. In one embodiment,antibodies directed to metadherin are administered to a patient in orderto prevent the metadherin molecules on the cancerous cells frommetastasizing. In one embodiment, the antibodies preferentially,specifically or selectively bind to metadherin. In one embodiment, theantibodies are directed to the lung-binding domain of metadherin, suchas the murine lung binding domain (SEQ ID NO: 3) or the correspondinghuman lung binding domain (SEQ ID NO: 17). In another embodiment,antisense RNA for metadherin is administered to the patient in order toreduce the expression of metadherin on the cancerous cells and thusreduce metastasis. In another embodiment, siRNA, directed to metadherin,is administered to a patient in order to reduce the level of expressionof metadherin in the cancerous cells. In one embodiment, the compound ismetadherin, a variant of metadherin, or a fragment of metadherin. In oneembodiment, the compound is a peptide derived from metadherin, or apeptidomimetic thereof.

Functional fragments of metadherin, methods of making the functionalfragments, and methods of using the functional fragments are alsodescribed below. In one embodiment, the functional fragments of themetadherin protein are used as homing agents in order to deliver adesired substance to the lungs. In one preferred embodiment, thefunctional fragment is a lung-binding domain, such as shown in SEQ IDNO: 3 or SEQ ID NO: 17. In another embodiment the functional fragment isa variant of the lung-binding domain. The functional fragments may beexpressed on cells; and the cells thereby become specifically localizedto the lung tissue. The possible lung-homing domain of metadherin isdisplayed in FIG. 1A. FIG. 1B displays the predicted hydrophobicity ofthe protein. FIG. 1C provides additional structural informationconcerning the metadherin protein. “TM” denotes the location of theputative transmembrane domain. The numbers denote the position of aminoacids in the metadherin protein.

In another embodiment, the functional fragments are attached to aprotein or drug of interest. In a more preferred embodiment, thesefragments, which are attached to a desired substance, are administeredto a patient and the fragments home to the lung tissue, delivering theprotein or drug of interest primarily to the lung tissues. In oneembodiment, the lung binding domain has the amino acid sequence suppliedin SEQ ID NO: 3 or SEQ ID NO: 17. In one embodiment, the compound orcompositions may be administered intravenously.

In an alternative embodiment, functional fragments of metadherin areused to compete with and block the metadherin receptors that reside onlung tissue. In a preferred embodiment, the functional fragmentscomprise the lung-binding domain of metadherin, as shown in SEQ ID NO: 3or SEQ ID NO: 17. In another preferred embodiment, the functionalfragments are variants of metadherin that prevent metastasis by bindingto the metadherin receptor.

In one embodiment, metadherin functional fragments are used to block thespread of a cancer. The functional fragments described above, whichprevent the association of metastatic cells with the lung tissue, areadministered to the patient's lung tissue where they can bind to themetadherin receptors.

In some embodiments, a method for diagnosing metastatic cancer in apatient is supplied. In some of these embodiments, a patient having atissue suspected to be at risk for metastatic spread of a cancer isselected. A determination is then made whether the tissue that is atrisk of metastasis is expressing a higher level of metadherin thannormal tissue. A higher expression level of metadherin is indicative ofmetastatic cancer. In other embodiments, binding agents that bind tometadherin are used as a diagnostic to detect the presence of cancer.The localization of a metadherin binding agent at a specific location ina tissue can indicate that the tissue has a possible cancerous area. Insome embodiments, the binding agent is a small peptide that binds tometadherin. In another embodiment, the binding agent is a receptor formetadherin. In other embodiments, the level of metadherin expression inthe tissue is examined by applying an antibody that preferentially bindsmetadherin. Preferably, the antibody is labeled with a radioactive orcolorometric label so that it can be easily detected. It should be notedthat this method can be performed on a tissue in vivo or in vitro. Whileany patient may benefit from this examination, there are options fornarrowing which patients to test. In one embodiment, the selection maybe done by family history. In another embodiment it may be determined bya correlation between cancers and risk of cancer spread, any riskgreater than 10% may be considered to result in a patient at risk. In analternative embodiment, any risk greater than 1% may be considered “atrisk.” As will be appreciated by one of skill in the art, the damagingeffects of the cancer may also be a factor to consider.

In one preferred embodiment, a phage expression library of cDNAs frommetastatic breast carcinoma is used to identify protein domains thatbind to the vasculature of the lung, a frequent site of breast cancermetastasis.

DEFINITIONS

The term “polynucleotide(s),” as used herein, means a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesand includes DNA and corresponding RNA molecules, including HnRNA andmRNA molecules, both sense and anti-sense strands, and comprehends cDNA,genomic DNA and recombinant DNA, as well as wholly or partiallysynthesized polynucleotides. An HnRNA molecule contains introns andcorresponds to a polynucleotide in a generally one-to-one manner. AnmRNA molecule corresponds to an HnRNA and polynucleotide from which theintrons have been excised. A polynucleotide may consist of an entiregene, or any portion thereof. Operable anti-sense polynucleotides maycomprise a fragment of the corresponding polynucleotide, and thedefinition of “polynucleotide” therefore includes all such operableanti-sense fragments.

The compositions and methods of the present invention also encompassvariants of the above polypeptides and polynucleotides. Such variantsinclude, but are not limited to, naturally occurring allelic variants ofthe inventive sequences.

A metadherin “variant,” as used herein, is a polypeptide,polynucleotide, or molecule that differs from the recited polypeptide orpolynucleotide, but only such that the activity of the metadherinprotein is not detrimentally altered. In a preferred embodiment, theability of the metadherin variant to bind to the metadherin receptor(s)is not detrimentally altered. In one preferred embodiment, the variantmetadherin molecule comprises or encodes for at least amino acids378-440 of the metadherin protein, but not the entire protein sequencein SEQ ID NO: 1. In another preferred embodiment, variant metadherinsdiffer from the wild-type sequence by substitution, deletion or additionof five amino acids or fewer. Such variants may generally be identifiedby modifying one of the above polypeptide sequences, and evaluating themetastasis preventing properties of the modified polypeptide using, forexample, the representative procedures described herein. Polypeptidevariants preferably exhibit at least about 70%, more preferably at leastabout 90%, more preferably at least about 95%, and most preferably atleast about 99% identity (determined as described below) to theidentified polypeptides. In a further preferred embodiment, the variantdiffers only in conservative substitutions and/or modifications.

As used herein, a “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also contain other modifications, including the deletion oraddition of amino acids, the alterations have minimal effect on thelung-binding domain. In a preferred embodiment, the alterations haveminimal influence on the metastasis preventing properties, secondarystructure and hydropathic nature of the metadherin polypeptide. Forexample, a metadherin polypeptide may be conjugated to a signal (orleader) sequence at the N-terminal end of the protein thatco-translationally or post-translationally directs transfer of theprotein. The metadherin polypeptide may also be conjugated to a linkeror other sequence for ease of synthesis, purification or identificationof the metadherin polypeptide (e.g., poly-H is), or to enhance bindingof the metadherin polypeptide to a solid support. For example, ametadherin polypeptide may be conjugated to an immunoglobulin Fc region.

A nucleotide “variant” is a sequence that differs from the recitednucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications may be readily introducedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis as taught, for example, by Adelman et al.(DNA, 2:183, 1983). Nucleotide variants may be naturally occurringallelic variants, or non-naturally occurring variants. Variantnucleotide sequences preferably exhibit at least about 70%, 75%, 80%,85%, 90% and most preferably at least about 95% identity (determined asdescribed below) to the recited sequence.

The functional fragments of the peptides provided by in one preferredembodiment include variants that are encoded by DNA sequences which aresubstantially homologous to one or more of the DNA sequencesspecifically recited herein. “Substantial homology,” as used herein,refers to DNA sequences that are capable of hybridizing under moderatelystringent conditions. Suitable moderately stringent conditions includeprewashing in a solution of 5×SSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0);hybridizing at 50° C.-65° C., 5×SSC, overnight or, in the event ofcross-species homology, at 45° C. with 0.5×SSC; followed by washingtwice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. Such hybridizing DNA sequences are also within thescope of this invention, as are nucleotide sequences that, due to codedegeneracy, encode an metadherin polypeptide that is encoded by ahybridizing DNA sequence.

In alternative embodiment, a homolog is one that can be aligned via aBLAST alignment and is as homologous to metadherin as one of thesequences already aligned in FIG. 2. In a preferred embodiment, the newsequence is a homolog if, following alignment of the sequence with themetadherin sequence, there are fewer variations between the potentialhomolog and metadherin than there are between the metadherin sequencedescribed herein and all of those listed in FIG. 2. In one embodimentthe metadherin is a human metadherin. In an alternative embodiment, themetadherin is a mouse metadherin. As will be appreciated by one of skillin the art, FIG. 2, and similar figures, may also be used to identifyfunctional domains of the metadherin protein. In one embodiment, domainsof the protein that are homologous across species are important domainsand may be useful for alternative “lung-binding” domains or othersignaling functions. In a more preferred embodiment, domains that areidentical across organisms in different classes are important domains.Thus, by this type of comparison, one may easily identify alternativefunctional domains of metadherin, of which the lung-binding domain isone example. In one embodiment, domains that are as similar as the foursequences presented in FIG. 2 are to one another, over the lung-bindingdomain, are considered homologous, and thus potential functionalfragments. One may then use the additional methods disclosed in thepresent specification to readily test the selected domains forfunctional activity.

Two nucleotide or polypeptide sequences are said to be “identical” ifthe sequence of nucleotides or amino acid residues in the two sequencesis the same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

In one embodiment, alignment of sequences for comparison may beconducted using the Megalign program in the Lasergene suite ofbioinformatics software (DNASTAR, Inc., Madison, Wis.), using defaultparameters. This program embodies several alignment schemes described inthe following references: Dayhoff, M. O. (1978) A model of evolutionarychange in proteins—Matrices for detecting distant relationships. InDayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp.345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp.626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,Calif.; Higgins, D. G. and Sharp, P. M. (1989) Fast and sensitivemultiple sequence alignments on a microcomputer CABIOS 5:151-153; Myers,E. W. and Muller W. (1988) Optimal alignments in linear space CABIOS4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M.(1987) The neighbor joining method. A new method for reconstructingphylogenetic trees Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. andSokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice ofNumerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J.and Lipman, D. J. (1983) Rapid similarity searches of nucleic acid andprotein data banks Proc. Natl. Acad, Sci. USA 80:726-730. There are manyoptions for alignment another option would be that offered by the ncbiwebpage, bl2seq.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e. the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Functional metadherin protein or metadherin fragments described hereinmay be created in any number of ways. They may be isolated from tissue,as described in the Examples. Alternatively, they may also be generatedby synthetic or recombinant means. Synthetic polypeptides having fewerthan about 100 amino acids, and generally fewer than about 50 aminoacids, may be generated using techniques well known to those of ordinaryskill in the art. For example, such polypeptides may be synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain (see, for example,Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963). Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

Alternatively, any of the above polypeptides may be producedrecombinantly by inserting a DNA sequence that encodes the polypeptideinto an expression vector and expressing the protein in an appropriatehost. Any of a variety of expression vectors known to those of ordinaryskill in the art may be employed to express recombinant polypeptides ofthis invention. Expression may be achieved in any appropriate host cellthat has been transformed or transfected with an expression vectorcontaining a polynucleotide that encodes a recombinant polypeptide.Suitable host cells include prokaryotes, yeast and higher eukaryoticcells. Preferably, the host cells employed are E. coli, yeast or amammalian cell line, such as CHO cells. The DNA sequences expressed inthis manner may encode naturally occurring polypeptides, portions ofnaturally occurring polypeptides, or other variants thereof.

Another embodiment includes mimetics of the metadherin protein. In amore preferred embodiment, the mimetic is of the lung-binding domain ofthe protein. By “mimetic,” it is meant that the functional structure ofthe lung-binding domain, is the same as the metadherin lung-bindingdomain. Mimetics may be determined by the same process that the originalmetadherin protein was discovered, as described in the presentspecification. Alternatively, mimetics may be created by sequenceanalysis and structural predictions, wherein predicted, or actual,protein structures for the metadherin lung-binding protein are used as ablueprint to create alternative mimetics of the lung-binding domain.Alternatively, the mimetics may not be structurally similar, but areonly functionally similar and may comprise small molecule drugs.

In one embodiment, the mimetics are small molecules and are functionallysimilar to the lung-binding domain of metadherin. Given the presentdisclosure, one of skill in the art could determine whether a particularsmall molecule was a mimetic of the lung-binding domain, simply byrepeating the procedure described herein and determining if the smallmolecule either bound in a similar fashion as the lung-binding domain,or if the mimetic had the same effect on metastasis, as the lung-bindingdomain. In one embodiment, these small molecules that mimic the activityof metadherin may be used to treat a patient, just as the lung-bindingdomain itself, as discussed herein, may be used to treat a patient. Inan alternative embodiment, the small molecules that mimic the activityof metadherin may be used as a diagnostic aid, in a manner similar tohow the lung-binding portion of metadherin can be used to diagnosecancer, as described herein.

In one embodiment, the mimetics are peptides or peptidomimetics,including bifunctional peptides, multivalent peptides andpeptidomimetics, and homing peptides and peptidomimetics discussedfurther below. As used herein, the term “peptide” is used broadly tomean peptides, proteins, fragments of proteins, and the like. The term“peptidomimetic,” as used herein, means a peptide-like molecule that hasthe activity of the peptide upon which it is structurally based. In oneembodiment, such peptidomimetics include chemically modified peptides,peptide like molecules containing non naturally occurring amino acids,and peptoids and have an activity such as selective homing activity ofthe peptide upon which the peptidomimetic is derived (see, for example,Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's MedicinalChemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff, John Wiley & Sons1995), pages 803 861).

A variety of peptidomimetics are known in the art including, as a matterof example, peptide like molecules which contain a constrained aminoacid, a nonpeptide component that mimics peptide secondary structure, oran amide bond isostere. Another example is a peptidomimetic thatcontains a constrained, non naturally occurring amino acid, for example,an α methylated amino acid; α,α dialkylglycine or α aminocycloalkanecarboxylic acid; an Nα Cα cyclized amino acid; an Nα methylated aminoacid; a β or γ amino cycloalkane carboxylic acid; an α,β unsaturatedamino acid; a β,β dimethyl or β methyl amino acid; a β substituted 2,3methano amino acid; an N Cδ or Cα Cδ cyclized amino acid; a substitutedproline or another amino acid mimetic. Another example is apeptidomimetic that mimics peptide secondary structure, for example, anon peptidic β turn mimic; γ turn mimic; mimic of β sheet structure; ormimic of helical structure, each of which is well known in the art.Another example is a peptidomimetic that can also be a peptide likemolecule that comprises, for example, an amide bond isostere such as aretro inverso modification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans olefin or fluoroolefin bond; 1,5 disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. As an example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptide of theinvention, as well as potential geometrical and chemical complementarityto a target molecule. Where no crystal structure of a peptide of theinvention or a target molecule that binds the peptide is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Information Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of a peptide of the invention, forexample, with activity in selectively binding to metadherin, variantsthereof or the metadherin receptor.

In one embodiment, an isolated peptide or peptidomimetic, or a homingmolecule, as will be discussed further below, can be cyclic or otherwiseconformationally constrained. As used herein, a “conformationallyconstrained” molecule, such as a peptide or peptidomimetic, is one inwhich the three dimensional structure is maintained substantially in onespatial arrangement over time. Conformationally constrained moleculescan have improved properties such as increased affinity, metabolicstability, membrane permeability or solubility. Methods ofconformational constraint are well known in the art and includecyclization as discussed further below.

As used herein in reference to a peptide or peptidomimetic, the term“cyclic” means a structure including an intramolecular bond between twononadjacent amino acids or amino acid analogues. The cyclization can beeffected through a covalent or noncovalent bond. Intramolecular bondsinclude, but are not limited to, backbone to backbone, side chain tobackbone and side chain to side chain bonds. A preferred method ofcyclization is through formation of a disulfide bond between the sidechains of nonadjacent amino acids or amino acid analogs. Residuescapable of forming a disulfide bond include, for example, cysteine(Cys), penicillamine (Pen), β,β pentamethylene cysteine (Pmc), β,βpentamethylene β mercaptopropionic acid (Pmp) and functional equivalentsthereof.

A peptide or peptidomimetic also can cyclize, for example, via a lactambond, which can utilize a side chain group of one amino acid or analogthereof to form a covalent attachment to the N terminal amine of theamino terminal residue. Residues capable of forming a lactam bondinclude aspartic acid (Asp), glutamic acid (Glu), lysine (Lys),ornithine (Orn), α,β diamino-propionic acid, γ amino adipic acid (Adp)and M (aminomethyl)benzoic acid (Mamb). Cyclization additionally can beeffected, for example, through the formation of a lysinonorleucine bondbetween lysine (Lys) and leucine (Leu) residues or a dityrosine bondbetween two tyrosine (Tyr) residues. One of ordinary skill in the artunderstands that these and other bonds can be included in a cyclicpeptide or peptidomimetic of the invention.

In another embodiment, the present invention provides for metadherin orvariants thereof, to be associated with and direct the localization of adesired substance. The “desired substance” can be any anything thatassociates with the metadherin protein. In one preferred embodiment,fusion proteins comprising a metadherin and a second polypeptide, thedesired substance, are linked together through covalent bonds. Inanother non-limiting example, cells that are artificially induced toexpress metadherin on their surface are the desired substance, that areassociated with metadherin, by expressing the metadherin on their cellsurface. In another embodiment, nanoparticles may be a desired substanceattached to the metadherin protein or variant. In another embodiment,nanodevices may be a desired substance attached to the metadherinprotein or variant. The methods of making and using the particles anddevices is well known in the art. For a review of this technology, seeErkki Ruoslahti, Cancer Cell, August 2002, 97-98.

These particles and devices, connected to the metadherin protein orvariant thereof, will then be delivered to the site of interest, by themetadherin protein or variant of this embodiment. For example, asdescribed by Hood et al., (Science, 296:2404-2407 (2002)), a molecule,in this instance a metadherin or variant thereof, may be added to theexternal surface of the nanoparticle, the result being that wherever themolecule would naturally be directed to, the entire nanoparticle will bedirected to and result in the delivery of the nanoparticle's cargo tothose localized cells. The cargo may be any compound, for example, itcan be protein based, DNA based, RNA based, a binding agent, atherapeutic agent, a diagnostic agent, or any other number ofpossibilities as discussed herein or as will be appreciated by one ofskill in the art.

In another embodiment, viruses may be the desired substance associatedwith a metadherin protein or variant thereof. In one embodiment, thevirus is a vaccinia or other pox virus, retrovirus, or adenovirus. Theuse of viruses to deliver genetic material for gene therapy is wellknown in the art, for example, see Panicali et al., (U.S. Pat. No.5,656,465, Issued Aug. 12, 1997), herein incorporated in its entirety byreference. One embodiment involves metadherin being cloned into a phagevector such as fuse 5 (for example, as described in Ruoslahti et al.,U.S. Pat. No. 6,610,651, Issued Aug. 26, 2003, herein incorporated inits entirety by reference), wherein, upon expression, the encodedpeptide is expressed as a fusion protein on the surface of the phage,thus directing the localization of the phage to areas with metadherinreceptors. In one embodiment, the viruses contain material to allow forgene therapy. The material may either help or kill the diseased cells.In another embodiment, the viruses contain material to allow for thediagnostic imaging of the tumors. Since the level of metadherin willindicate the risk of cancer, the materials need not be involved indetecting the cancer and may simply be a detectable probe, such as greenfluorescent protein. However, in another embodiment, the material in thevirus may also serve to actually detect the presence of differentmarkers on cells.

In some embodiments, a “linking element” is used to attach themetadherin or metadherin variant to the desired substance. One of skillin the art will recognize that there are many alternatives by which onecan connect two molecules. A linking element may be a complex moleculewith particular desired characteristics, such as light cleavable bonds,or it could be as simple as a disulfide bond. When another molecule ordesired substance is associated with a metadherin or metadherin variant,it results in the formation of a conjugate. The term “conjugate” ismeant to denote the association of both the desired substance andanother compound or molecule. In one embodiment, it is meant to denote adesired substance associated with metadherin or a metadherin variant. Inanother embodiment, it is meant to denote a desired substance associatedwith an antibody to metadherin or a metadherin variant. In anotherembodiment, it is meant to denote a desired substance associated with abinding agent of metadherin. Conjugates may be additionally defined asdescribed in this specification, or as would be understood by one ofskill in the art.

In one embodiment, a DNA sequence encoding a fusion protein of thepresent invention is constructed using known recombinant DNA techniquesto assemble separate DNA sequences encoding the metadherin and secondpolypeptides into an appropriate expression vector. The 3′ end of a DNAsequence encoding the metadherin is ligated, with or without a peptidelinker, to the 5′ end of a DNA sequence encoding the second polypeptideso that the reading frames of the sequences are in phase to permit mRNAtranslation of the two DNA sequences into a single fusion protein thatretains the biological activity of both the metadherin and the secondpolypeptides. As appreciated by one of skill in the art, only a fractionof the metadherin molecule needs to be included in the fission protein.In a preferred embodiment, only amino acids 378-440 of SEQ ID NO: 1 areused.

A peptide linker sequence may be employed to separate the first and thesecond polypeptides by a distance sufficient to ensure that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may be from 1 to about 50 amino acids in length.Peptide sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptide. Similarly, stop codonsrequire to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide. Themetadherin polypeptide need not be the first in the nucleotide sequence.

In one embodiment, a second polypeptide, encoded by a secondpolynucleotide is a means to attach desired substances to metadherin,thus allowing metadherin to direct desired substances to the lungs, evenif the desired substances are not nucleotide based. The secondpolypeptide of this embodiment may either have its own binding abilityor may simply be a molecule that is readily bound by other bindingentities.

In an alternative embodiment, there is no linker or secondpolynucleotide involved; however, there is a desired substance. Thedesired substance has the ability to bind or associate itself withmetadherin polypeptide. In a further preferred embodiment, the method bywhich the desired substance binds itself to metadherin does not blockthe lung-binding domain of metadherin. In another preferred embodiment,residues 378-440 of metadherin are not blocked by the association of thedesired substance with metadherin. By “lung-binding domain,” all that ismeant is the structure by which metadherin binds to its receptors.Depending upon the context, this structure may be a nucleic acidstructure, the amino acid structure or a non protein structure whichresembles the three dimensional shape of the lung-binding domain.“Lung-binding domain” does not mean that the domain only binds toreceptors located in the lung. In one embodiment, the “lung-bindingdomain” may bind to similar metadherin receptors located in otherorgans. In one embodiment, the lung-binding domain may bind to similarreceptors in the liver. In one embodiment, the lung-binding domain willalso bind to different receptors in the liver. As will be appreciated byone of skill in the art, all of the embodiments herein, which apply tothe use of metadherin with the lungs, may also be applied to otherorgans that also express a effective expression level of a receptor thatbinds to metadherin. For example, if metadherin or variants thereof,also bind to receptors in the liver, then the metadherin or variantsthereof can be used as a diagnostic, as described herein for lungdiagnosis, or the metadherin or variants thereof, can be used to stopthe spread of a cancer to the liver, as described herein for stoppingbreast cancer. One of skill in the art will appreciate the changes thatmust be made for localization to a particular location in the body.

In one embodiment, the desired substance incorporated into a conjugateof the invention is a therapeutic agent. As used herein, the term“therapeutic agent” means a molecule that has one or more biologicalactivities in a normal or pathologic tissue. A variety of therapeuticagents can be included in a conjugate of the invention. In oneembodiment, a conjugate of metadherin, or variant thereof, contains acancer chemotherapeutic agent. As used herein, a “cancerchemotherapeutic agent” is a chemical agent that inhibits theproliferation, growth, life span or metastatic activity of cancer cells.Such a cancer chemotherapeutic agent can be, without limitation, ataxane such as docetaxel; an anthracyclin such as doxorubicin; analkylating agent; a vinca alkaloid; an anti metabolite; a platinum agentsuch as cisplatin or carboplatin; a steroid such as methotrexate; anantibiotic such as adriamycin; a isofamide; or a selective estrogenreceptor modulator; an antibody such as trastuzumab.

In one embodiment, taxanes are chemotherapeutic agents useful in theconjugates of the invention. Useful taxanes include, without limitation,docetaxel (Taxotere; Aventis Pharmaceuticals, Inc.; Parsippany, N.J.)and paclitaxel (Taxol; Bristol Myers Squibb; Princeton, N.J.). See, forexample, Chan et al., J. Clin. Oncol. 17:2341-2354 (1999).

In one embodiment, a cancer chemotherapeutic agent useful in a conjugateof the invention is an anthracyclin such as doxorubicin, idarubicin ordaunorubicin. Doxorubicin is a commonly used cancer chemotherapeuticagent and can be useful, for example, for treating breast cancer(Stewart and Ratain, In: “Cancer: Principles and practice of oncology”5th ed., chap. 19 (eds. DeVita, Jr., et al.; J. P. Lippincott 1997);Harris et al., In “Cancer: Principles and practice of oncology,” supra,1997). In addition, doxorubicin has anti angiogenic activity (Folkman,Nature Biotechnology 15:510 (1997); Steiner, In “Angiogenesis: Keyprinciples Science, technology and medicine,” pp. 449-454 (eds. Steineret al.; Birkhauser Verlag, 1992)), which can contribute to itseffectiveness in treating cancer.

In one embodiment, an alkylating agent such as melphalan or chlorambucilis a cancer chemotherapeutic agent useful in a conjugate of theinvention. Similarly, a vinca alkaloid, such as, vindesine, vinblastineor vinorelbine; or an antimetabolite, such as, 5 fluorouracil, 5fluorouridine or a derivative thereof can be a cancer chemotherapeuticagent useful in a conjugate of the invention.

In one embodiment, a platinum agent is a cancer chemotherapeutic agentuseful in the conjugates of the invention. Such a platinum agent can be,for example, cisplatin or carboplatin as described, for example, inCrown, Seminars in Oncol. 28:28-37 (2001). Other cancer chemotherapeuticagents useful in a conjugate of the invention include, withoutlimitation, methotrexate, mitomycin C, adriamycin, ifosfamide andansamycins.

In one embodiment, a cancer chemotherapeutic agent for treatment ofbreast cancer, or other hormonally dependent cancers, is an agent thatantagonizes the effect of estrogen, such as a selective estrogenreceptor modulator or an anti estrogen. The selective estrogen receptormodulator, tamoxifen, is a cancer chemotherapeutic agent that can beused in a conjugate of the invention for treatment of breast cancer(Fisher et al., J. Natl. Cancer Instit. 90:1371 1388 (1998)).

In one embodiment, a therapeutic agent useful in a conjugate of theinvention is an antibody such as a humanized monoclonal antibody. As anon-limiting example, the anti epidermal growth factor receptor 2 (HER2)antibody, trastuzumab (Herceptin; Genentech, South San Francisco,Calif.) is a therapeutic agent useful in a conjugate of the inventionfor treating HER2/neu overexpressing breast cancers (White et al., Annu.Rev. Med. 52:125 141 (2001)).

In one embodiment, a therapeutic agent useful in the invention is acytotoxic agent. A “cytotoxic agent,” as used herein, is any moleculethat directly or indirectly promotes cell death. Cytotoxic agents usefulin the invention include, without limitation, small molecules,polypeptides, peptides, peptidomimetics, nucleic acid molecules, cellsand viruses. As non limiting examples, cytotoxic agents useful in theinvention include cytotoxic small molecules such as doxorubicin,docetaxel or trastuzumab; antimicrobial peptides such as those describedfurther below; pro-apoptotic polypeptides such as caspases and toxins,for example, caspase 8; diphtheria toxin A chain, Pseudomonas exotoxinA, cholera toxin, ligand fusion toxins such as DAB389EGF, ricinuscommunis toxin (ricin); and cytotoxic cells such as cytotoxic T cells.See, for example, Martin et al., Cancer Res. 60:3218 3224 (2000);Kreitman and Pastan, Blood 90:252 259 (1997); Allam et al., Cancer Res.57:2615 2618 (1997); and Osborne and Coronado Heinsohn, Cancer J. Sci.Am. 2:175 (1996). One skilled in the art understands that these andadditional cytotoxic agents described herein or known in the art can beuseful in the conjugates and methods of the invention.

In one embodiment, a therapeutic agent is a therapeutic polypeptide. Asused herein, a “therapeutic polypeptide” is any polypeptide with abiologically useful function. Therapeutic polypeptides useful in theinvention include, without limitation, cytokines, antibodies, cytotoxicpolypeptides, pro apoptotic polypeptides, and anti angiogenicpolypeptides. As nonlimiting examples, a therapeutic polypeptide usefulin the invention can be a cytokine such as tumor necrosis factor α (TNFα), tumor necrosis factor β (TNF β), granulocyte macrophage colonystimulating factor (GM CSF), granulocyte colony stimulating factor (GCSF), interferon α (IFN α); interferon γ (IFN γ), interleukin I (IL 1),interleukin 2 (IL 2), interleukin 3 (IL 3), interleukin 4 (IL 4),interleukin 6 (IL 6), interleukin 7 (IL 7), interleukin 10 (IL 10),interleukin 12 (IL 12), lymphotactin (LTN) or dendritic cell chemokine 1(DC CK1); an anti HER2 antibody or fragment thereof; a cytotoxicpolypeptide including a toxin or caspase, for example, diphtheria toxinA chain, Pseudomonas exotoxin A, cholera toxin, a ligand fusion toxinsuch as DAB389EGF or ricin; or an anti angiogenic polypeptide such asangiostatin, endostatin, thrombospondin, platelet factor 4; anastellin;or one of those described further herein or known in the art (seebelow). It is understood by one of skill in the art that these and otherpolypeptides with biological activity can be a “therapeutic polypeptide”useful in the invention.

The term “compound” is meant to denote a broad variety of substances andcan encompass, for example, toxins, agents, cytotoxic agents, siRNA,therapeutic agents, antibodies, binding agents, metadherin receptorproteins, metadherin receptor nucleic acid sequence, metadherin aminoacid sequence and metadherin nucleic acid sequences, metadherinlung-binding domain, and variants thereof. The particular group orsubgroup will depend upon how the term is used.

The term “agent” can denote the presence of a molecule that is to beassociated with a metadherin protein or nucleotide sequence. However, insome situations, the term agent also encompasses the protein or nucleicacid sequences of metadherin.

A “test compound” is a compound that can be administered either in vivo,in vitro, in silico, or by another manner, so as to be present whenmetadherin binds to its receptor. Test compounds can either block theinteraction between metadherin and its receptor (either partially orcompletely), or not interfere with the interaction of metadherin and itsreceptor. Test compounds can include the above agents, antibodies,siRNA, therapeutics, etc. Successful test compounds will be those thatinhibit the functional interaction between metadherin and its receptor.Thus, binding may still occur, but the binding will not be sufficient toallow metadherin to function in metastasis. Alternatively, a successfultest compound may effectively prevent the binding of metadherin to itsreceptor under in vivo conditions and concentrations. Alternatively,binding simply may not occur because there is no or a reduced amount ofmetadherin which can bind. Examples of successful test compositions caninclude variants of the lung-binding domain, siRNAs, peptidomimetics,and various agents and therapeutics, for example.

Metadherin

As described in the below and in the examples, a protein thatselectively targeted phage to lung tissue was discovered and named“metadherin.” A nucleic acid sequence of the protein is shown in SEQ IDNO: 2 (murine) and 16 (human). An amino acid sequence of the protein isshown in SEQ ID NO: 1 (murine) and 13 (human). FIG. 1A displays ametadherin sequence and identifies the putative lung-homing domain inthe protein. FIG. 1B demonstrates a hydrophobicity plot of the protein,which suggests various structural features of the protein. By insertinga myc epitope tag into the protein (e.g., as shown in FIG. 1C) theprotein was found to have an extra-cellular domain involved in celladhesion. This domain not only targeted phage to lung tissue, but wasalso, found to localize cells that were over-expressing metadherin tothe lung tissue.

High expression of metadherin was found in cultured tumor cells and inboth experimental and clinical breast cancers. The high expression ofmetadherin was selective for cancers in that the expression in normalbreast tissue and in other normal tissues was low, as detected withmetadherin-specific antibodies.

Immunostaining revealed that metadherin was highly expressed in breastcancer tissue and breast tumor xenografts. As illustrated below,antibodies reactive to a lung-homing domain (SEQ ID NO: 3) of metadherininhibited experimental lung metastasis, indicating that metadherinexpressed on the surface of tumor cells mediated the localization, andpossibly the growth, at the metastatic site. Additionally,siRNA-mediated inhibition of metadherin expression in breast cancercells likewise was discovered to inhibit experimental lung metastasis.

Metadherin appears to detect and target a specific marker of lungvasculature. Phage displaying metadherin accumulated in lungvasculature, suggesting that among the various vascular beds, itprimarily binds to lung endothelium. The ability of metadherin phage tospecifically target lung vasculature suggests that among the variousvascular beds, the metadherin receptor is primarily expressed on lungendothelium.

As such, it has been discovered that metadherin appears to play animportant role in cancer metastasis and that compounds and methods thatalter metadherin availability can thereby be used to alter and reducecancer metastasis and the risk thereof.

The importance of metadherin in tumor cell metastasis is not be limitedto breast cancer. By use of SAGEmap (Lal, A., et al., Cancer Res 59:5403-5407 (1999); Lash, A. E., et al., Genome Res 10: 1051-1060 (2000)),a component of The Cancer Genome Anatomy Project at the National Centerfor Biotechnology Information, it was determined that metadherin mRNA issignificantly over-expressed in cancers of the brain and prostate(P<0.05). This suggests that metadherin might also play a role in themetastasis of these cancers as well. Thus, the general methods andcompositions described in the present specification may be used, notonly for breast cancer, but also for any cancer with elevated levels ofmetadherin.

Metadherin is conserved among mammals, and with the BLAST algorithm(Altschul, S. F., et al., Nucleic Acids Res 25: 3389-3402 (1997)), itwas determined that there were additional mouse and humanmetadherin-like molecules in the GenBank™ databases.

In some embodiments, the metadherin protein, or variant thereof, can beexpressed in cells that then express metadherin on their surface andthus allow localization of the cells to the lung tissue. In oneembodiment, only the lung-binding domain of metadherin need be expressedon the cell to be delivered to the lungs. A simple method to create suchhoming cells is to make the cells express metadherin, the lung-bindingdomain of metadherin, or a chimera of metadherin on their cell surface,and then administer the cells to the patient. In an alternativeembodiment, the metadherin molecules are not expressed on the cells butare instead associated with the cells. While there are many ways ofassociating a peptide with a cell, a linker, which binds to themetadherin peptide and to a particular molecule on the cell is onecommon way of creating such an association. The linker can either bindto particular proteins or molecules on the cell, or it canindiscriminately bind to the cell membrane.

In other embodiments, metadherin or a variant or fragment thereof can beused to stop metastasis. Metadherin peptides can be administered to apatient with lung or breast cancer to stop or reduce the risk ofmetastasis. The metadherin proteins can be administered in a variety ofways, as discussed below, and can either be administered to the lungsdirectly or elsewhere, since the metadherin molecules will localize tothe lung tissue eventually on their own. The entire metadherin proteinneed not be administered, as any functional variant will also localizeto the lung. The metadherin, or variant thereof, may be administered inthe lung tissue in an amount sufficient to block a substantial amount ofthe metadherin receptors in the lung and thus prevent later metadherinmolecules from localizing in the lung tissue. Alternatively, themetadherin receptors or variants thereof may be added to the initialtumor, or lung tissue, in order to bind to the metadherin on thecancerous cells. This in turn prevents the metadherin on the cancerouscells from binding to the receptors on the lung tissue, therebypreventing tumor metastasis to the lungs.

In a preferred embodiment, a lung-binding domain of metadherin isadministered to the patient in order to reduce the chances of anynonspecific binding occurring between the fragment and unknown targetsin the patient. A “fragment” as used herein is a subset of a “variant.”In other words, a variant may include species that are not fragments. Ina preferred embodiment, only cell-free metadherin is administered to thelungs, so that the binding of metadherin to the surface of the lungtissue will have a minimal impact on any local tissue. The metadherin ormetadherin fragment will localize to the lungs where it will occupy themetadherin receptors present in the lungs and prevent cancer cells,which associate with the lung tissue via the metadherin receptors fromlocalizing to the lung, thus preventing metastasis. As will beappreciated by one of skill in the art, the peptide or full lengthprotein need not be administered as a peptide and can be administered innucleic acid form to later be expressed in vivo.

In an alternative embodiment, antisense RNA that binds and inhibitstranslation of metadherin is used to treat metastatic or potentiallymetastatic tumors. In a preferred embodiment, the RNA is anti-sense RNAthat prevents effective production of the metadherin protein. In analternative embodiment, small interfering RNAs (siRNAs) are used totreat metastatic tumors. The structure and activity of siRNA is reviewedby Bosher et al. (Nature Cell Biol. 2:E31, 2000) and C. P. Hunter (Curr.Biol. 9:R440, 1999). Such siRNA complexes comprise double-stranded RNAmolecules having metadherin sense and antisense polynucleotides thatspecifically inhibit translation of mRNA encoding metadherin. Kits andinstructions for using or creating siRNA are plentiful in the industryand are available at many companies. For example, Ambion (Austin, Tex.)makes several kits for in vitro siRNA production, such as the “SilencersiRNA Construction Kit.” As described below, the pSilencer 3.0-H1plasmid, and an Ambion vector for making siRNA, was used effectively toreduce metastatic breast cancer.

Metadherin, or metadherin variants, including fusion proteins, can bepresent within a pharmaceutical composition and/or a vaccine.Pharmaceutical compositions may comprise one or more polypeptides, eachof which may contain one or metadherins, and a physiologicallyacceptable carrier. Pharmaceutical compositions may also comprise smallmolecules, antibodies, peptides, polynucleotides, anti-sense RNA andsiRNA.

There can be situations where it is desired to have the body make itsown antibodies to metadherin, in order to constantly produce antibodiesto block metadherin binding. In such a case, one may use the metadherinmolecule, or variant thereof to make a vaccine to metadherin. Thevaccines may comprise one or more metadherins and a non-specific immuneresponse enhancer, wherein the non-specific immune response enhancer iscapable of eliciting or enhancing an immune response to a metadherin.Examples of non-specific-immune response enhancers include adjuvants,biodegradable microspheres (e.g., polylactic galactide) and liposomes(into which the polypeptide is incorporated). Pharmaceuticalcompositions and vaccines may also contain other epitopes of breasttumor antigens, either incorporated into a combination polypeptide(i.e., a single polypeptide that contains multiple epitopes) or presentwithin a separate polypeptide.

Alternatively, a pharmaceutical composition or vaccine may contain DNAencoding one or more metadherins, such that the metadherin is generatedin situ. In such pharmaceutical compositions and vaccines, the DNA maybe present within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter). Bacterial delivery systemsinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an epitope of a breast tumorcell antigen on its cell surface. In a preferred embodiment, the DNA maybe introduced using a viral expression system (e.g., vaccinia or otherpox virus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., PNAS86:317-321, 1989; Flexner et. al., Ann. N.Y. Acad. Sci. 569:86-103,1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112,4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627,1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzmanet al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in published PCTapplication WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993,reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNAmay be increased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells.

Routes and frequency of administration, as well as dosage, will varyfrom individual to individual and may parallel those currently beingused for other diseases. In general, the pharmaceutical compositions andvaccines may be administered by injection (e.g., intracutaneous,intramuscular, intravenous or subcutaneous), intranasally (e.g., byaspiration) or orally.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a lipid, a waxand/or a buffer. For oral administration, any of the above carriers or asolid carrier, such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, talcum, cellulose, glucose, sucrose, and/or magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticglycolide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

In a further embodiment, the embodiments above are combined in a kit inorder to administer either a treatment to stop metastasis, a test todiagnosis the presence or risk of a cancer spreading, or both. In onepreferred embodiment, the carriers and the metadherin variants, or thebinding agents described below, are combined to provide a pill form,which is then packaged so as to allow for regular dosage for theprevention of cancer metastasis. In one preferred embodiment, a kit forthe treatment to stop the metastasis of lung cancer would includemultiple embodiments of the invention. It could include a metadherinbased device to prevent metastasis, such as a fragment of metadherin,and a metadherin based device to detect the amount the amount of nativemetadherin remaining in the system, perhaps an antibody detection systemdirected against metadherin.

In a different embodiment, the conjugates and therapeutic agentsdiscussed above, and the binding agents discussed below, can be used inmethods of imaging tumor metastasis. In one embodiment, the bindingagents also block the binding of metadherin to its receptor.

The conjugates and agents can be useful for detecting the presence ofmetastasized tumor cells associated with a variety of tumors, includingbreast, ovarian, brain, colon, kidney, lung, liver, bladder and prostatetumors and melanomas. Following administration of an agent or conjugatecontaining a detectable agent, tumor metastasis is visualized. If theimage is positive for the presence of tumor metastasis, the tumor can beevaluated for further treatment. These results provide valuableinformation to the clinician with regard to the stage of development ofthe cancer and the presence or probability of metastasis.

In one embodiment, a method of imaging tumor lymphatic vasculature isprovided. The conjugate or agent administered contains a detectableagent that allows detection or visualization of vasculature in andaround tumors, for example in and around breast tumors. For in vivodiagnostic imaging of tumor metastasis, a peptide or other agent whichbinds to metadherin, variant thereof, or the metadherin receptor islinked to a detectable agent that, upon administration to the subject,is detectable external to the subject. In one embodiment, such adetectable agent may be, for example, a gamma ray emitting radionuclidesuch as indium 113, indium 115 or technetium 99; followingadministration to a subject, the conjugate or agent may be visualizedusing a solid scintillation detector.

A variety of detectable agents are useful in the methods of theinvention. As used herein, the term “detectable agent” refers to anymolecule that can be administered in vivo and subsequently detected. Inone embodiment, detectable agents useful in the imaging methods of theembodiment include, but are not limited to, radioactive agents andcolorometric agents. Exemplary radioactive agents include indium 111,technetium 99, carbon 11, and carbon 13. Fluorescent molecules useful inthe invention encompass, without limitation, fluorescein,allophycocyanin, phycoerythrin, rhodamine, and Texas red.

In another embodiment, the agents and conjugates used in this methodcomprise a mimic to the lung-binding domain of metadherin, as well asthe detectable agent.

In one embodiment, metadherin, or variants thereof, may be used togenerate binding agents, such as antibodies or fragments thereof. In apreferred embodiment, these binding agents are capable of detecting orpreventing the metastasis of human breast or lung tumors. Binding agentsof the present invention may generally be prepared using methods knownto those of ordinary skill in the art, including the representativeprocedures described herein. In one embodiment, binding agents arecapable of differentiating between patients with and without lung orbreast cancer, using the representative assays described herein. Inother words, antibodies or other binding agents that bind to metadherin,or a suitable variant thereof, will generate a signal indicating thepresence of lung or breast cancer. In a different embodiment, theemphasis of the binding agent is not in distinguishing between thepresence or absence of cancer, but in how efficiently the binding agentbinds to metadherin.

The ability of metadherin or metadherin variants to generate antibodiescapable of detecting tumors may generally be evaluated by raising one ormore antibodies against metadherin, or a metadherin variant, anddetermining the ability of the antibodies to detect tumors in patients.This determination may be made by assaying biological samples frompatients with and without cancer for the presence of metadherin that isbound by the generated antibodies. Such test assays may be performed,for example, using a representative procedure described below.Antibodies capable of detecting tumors by such procedures are considereduseful. In a further preferred embodiment, metadherin variants orfragments that generate antibodies capable of detecting at least 20% oftumors by such procedures are considered to be very useful in assays fordetecting metastatic human lung or breast tumors. Antibodies may be usedalone or in combination to improve sensitivity.

Metadherin may be used as a marker for diagnosing cancer or formonitoring disease progression in patients. In one embodiment, cancer ina patient may be diagnosed by evaluating a biological sample obtainedfrom the patient for the level of metadherin; relative to apredetermined cut-off value. As used herein, suitable “biologicalsamples” include blood, sera, urine, as well as the more traditionaltissue samples. In a preferred embodiment, the sample is a lung tissuesample.

The level of metadherin may be evaluated using any binding agent that isspecific for metadherin. A “binding agent,” in the context of theinvention, is any agent (such as a compound or a cell) that binds tometadherin. As used herein, “binding” refers to an association betweentwo separate molecules (each of which may be free (i.e., in solution) orpresent on the surface of a cell or a solid support), such that a“complex” is formed. Such a complex may be free or immobilized (eithercovalently or noncovalently) on a support material. The ability to bindmay generally be evaluated by determining a binding constant for theformation of the complex. The binding constant is the value obtainedwhen the concentration of the complex is divided by the product of thecomponent concentrations. In a preferred embodiment, two compounds aresaid to “bind” in the context of the present invention when the bindingconstant for complex formation exceeds about 10³ L/mol. The bindingconstant may be determined using methods well known to those of ordinaryskill in the art. Binding may either be covalent, such as with theformation of disulfide bonds, or it may be non-covalent.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome with or without a peptidecomponent, an RNA molecule or a peptide. The binding agents of thepresent embodiments need not bind exclusively to metadherin. In oneembodiment, the binding agents preferentially bind to metadherin. By“preferentially” all that is meant is that the binding agent binds tometadherin with a stronger preference than it binds to at least oneother target compound. Similarly, if a binding agent is “specific” formetadherin, all that is meant is that the binding agent binds tometadherin with greater specificity than it does to one other material.As will be appreciated by one of skill in the art, the degree ofspecificity required in any particular application of the binding agentswill depend upon many factors, including the other molecules to whichthe binding agent will be exposed.

In a preferred embodiment, the binding agent is an antibody, or abinding fragment thereof. Such antibodies may be polyclonal, ormonoclonal. In addition, the antibodies may be single chain, chimeric,CDR-grafted or humanized. Antibodies may be prepared by the methodsdescribed herein and by other methods well known to those of skill inthe art.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding partner to detect a molecule like metadherinin a sample. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment,the assay involves the use of binding partner immobilized on a solidsupport to bind to and remove the polypeptide from the remainder of thesample. The bound polypeptide may then be detected using a secondbinding partner that contains a reporter group. Suitable second bindingpartners include antibodies that bind to the binding partner/polypeptidecomplex. Alternatively, a competitive assay may be utilized, in which ametadherin is labeled with a reporter group and allowed to bind to theimmobilized binding partner after incubation of the binding partner withthe sample. The extent to which components of the sample inhibit thebinding of the labeled metadherin to the binding partner is indicativeof the reactivity of the sample with the immobilized binding partner.

The solid support may be any material known to those of ordinary skillin the art to which the antigen may be attached. For example, the solidsupport may be a test well in a microtiter plate or a nitrocellulose orother suitable membrane. Alternatively, the support may be a bead ordisc, such as glass, fiberglass, latex or a plastic material such aspolystyrene or polyvinylchloride. The support may also be a magneticparticle or a fiber optic sensor, such as those disclosed, for example,in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on thesolid support using a variety of techniques known to those of skill inthe art, which are amply described in the patent and scientificliterature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the antigen and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but in a preferred embodimentis typically between about 1 hour and about 1 day. In general,contacting a well of a plastic microtiter plate (such as polystyrene orpolyvinylchloride) with an amount of binding agent ranging from about 10ng to about 10 microgram, and preferably about 100 ng to about 1microgram, is sufficient to immobilize an adequate amount of bindingagent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides of metadherin, or fragments ofmetadherin, within the sample are allowed to bind to the immobilizedantibody. Unbound sample is then removed from the immobilizedmetadherin-antibody complexes and a second antibody (containing areporter group) capable of binding to a different site on metadherin isadded. The amount of second antibody that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or TWEEN 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and metadherin, or a fragment thereof, isallowed to bind to the antibody. The sample may be diluted with asuitable diluent, such as phosphate-buffered saline (PBS) prior toincubation. In general, an appropriate contact time is that period oftime that is sufficient to detect the presence of metadherin within asample obtained from an individual with cancer. In a preferredembodiment, the contact time is sufficient to achieve a level of bindingthat is at least about 95% of that achieved at equilibrium between boundand unbound metadherin. Those of ordinary skill in the art willrecognize that the time necessary to achieve equilibrium may be readilydetermined by assaying the level of binding that occurs over a period oftime. As a general guideline, at room temperature, an incubation time ofabout 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20®. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of antibody to reporter group may be achieved using standardmethods known to those of ordinary skill in the art.

The second antibody is then incubated with the immobilizedantibody-metadherin complex for an amount of time sufficient to detectthe bound metadherin. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound second antibody is then removed and bound second antibodyis detected using the reporter group. The method employed for detectingthe reporter group depends upon the nature of the reporter group. Forradioactive groups, scintillation counting or autoradiographic methodsare generally appropriate. Spectroscopic methods may be used to detectdyes, luminescent groups and fluorescent groups. Biotin may be detectedusing avidin, coupled to a different reporter group (commonly aradioactive or fluorescent group or an enzyme). Enzyme reporter groupsmay generally be detected by the addition of substrate (generally for aspecific period of time), followed by spectroscopic or other analysis ofthe reaction products.

To determine the risk of the spread of cancer in a patient, the signaldetected from the reporter group that remains bound to the solid supportis generally compared to a signal that corresponds to a predeterminedcut-off value. In one preferred embodiment, the cut-off value is theaverage mean signal obtained when the immobilized antibody is incubatedwith samples from patients without cancer. In another preferredembodiment, the cut-off value is determined by the amount of metadherinin the system of one that has cancer, but no spread of cancer hasoccurred. In one embodiment, when the sample generating the signal ishigher than the cut-off value, then there is a risk of metastasis ofcancer in that tissue. In a preferred embodiment, a sample generating asignal that is three standard deviations above the predetermined cut-offvalue is considered positive for a risk of spread of the cancer to thatorgan. In an alternate preferred embodiment, the cut-off value isdetermined using a Receiver Operator Curve, according to the method ofSackett et al., Clinical Epidemiology: A Basic Science for ClinicalMedicine, Little Brown and Co., 1985, p. 106-7. Briefly, in thisembodiment, the cut-off value may be determined from a plot of pairs oftrue positive rates (i.e., sensitivity) and false positive rates(100%-specificity) that correspond to each possible cut-off value forthe diagnostic test result. The cut-off value on the plot that is theclosest to the upper left-hand corner (i.e., the value that encloses thelargest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate. Ingeneral, a sample generating a signal that is higher than the cut-offvalue determined by this method is considered positive for cancer. In analternate preferred embodiment, the cut-off value is the average meansignal obtained when the immobilized antibody is incubated with samplesfrom patients with cancer. In this embodiment, the result from thesample examined will be as large or larger than the sample from theknown cancer.

In another embodiment, metadherin may be used as a marker for theprogression of lung or breast cancer. In this embodiment, assays asdescribed above for the diagnosis of cancer may be performed over time,and the change in the amount of metadherin evaluated. In general, lungor breast cancer is progressing in those patients in whom the level ofmetadherin detected by the binding agent increases over time. Incontrast, lung or breast cancer is not progressing when the level ofreactive metadherin either remains constant or decreases with time.

Metadherin, or variants thereof, may also, or alternatively, be used togenerate binding agents that are capable of inducing a change in thebinding characteristics of metadherin. Such binding agents would allowone to bind to and alter the binding characteristics of the metadherinpolypeptide that is already present on the cancerous cells. The resultbeing that the lung-binding domain of the cancerous cell wouldeffectively be neutralized and the cancer would not spread to othersections of the patient, particularly the lungs. In a preferredembodiment, the binding agent blocks the binding ability of metadherinto the metadherin receptor(s). In another preferred embodiment, thebinding agents bind to and block the lung-binding domain of metadherin.In an alternative embodiment, the binding agent may actually promote orstrengthen the binding of metadherin and its receptor. In order to beperfectly clear on terminology, such binding agents should be referredto as “enhancing binding agents,” rather than simply binding agents. Inone embodiment, the binding agents are small molecules that bind to andblock metadherin binding. In another embodiment, the binding agents arepeptides. In another embodiment, the binding agents are antibodies. Inone embodiment, the binding agents may be used to either treat orprevent the spread of a cancer. In another embodiment, the bindingagents may be used in diagnostic imaging methods, as described herein.

There are many ways to determine if a small molecule, peptide, protein,or antibody has an effect on the binding characteristics of metadherin.In general, the methods described previously concerning detection ofmetadherin will be useful in observing if the binding characteristics ofmetadherin have been altered by the binding agent. An example of onesuch binding agent is demonstrated in the Examples below. In a preferredembodiment, the metadherin protein and possible binding agents are firstallowed to bind together to form a complex under conditions that wouldbe appropriate for the desired properties of the final binding agent.This complex is then exposed to metadherin receptors. There are avariety of ways to do this. In one preferred embodiment, the complex isexpressed on the surface of a cell and then the cell is injected into ananimal to see if it localizes to the lung tissue, in particular, to themetadherin receptors. If the presence of the binding agent reduces thenumber of cells that are localized to the lungs, then the binding agentwill be an effective binding agent that modulates the binding ability ofmetadherin.

In an alternative preferred embodiment, a similar process willdemonstrate if an agent is an effective means for modulating metadherinactivity, in other words, is the agent an activity modulating agent.This can be tested for by determining if cancerous cells, expressingmetadherin, can still localize to the lungs after or during exposure tothe possible activity modulating agent in question.

In one embodiment, a method for screening compounds (e.g., testcompounds) that alter tumor metastasis is provided. The embodimentcomprises selecting a first test compound, contacting the first testcompound in the presence of a metadherin polypeptide, variant, orfragment thereof, and the metadherin receptor, and then determiningwhether the first compound affects the binding of the metadherinpolypeptide, variant or fragment to the metadherin receptor. In analternative embodiment, the effect to be observed may be to determine iftumor metastasis occurs, rather than simply looking for binding of thecompound to the metadherin receptor. In one embodiment, the metadherinreceptor is located on the lung vasculature.

In a further embodiment, it may be desired to distinguish between agentsin the above embodiments, that is, to determine if the agent is abinding agent, reducing metadherin localization by blocking the bindingof metadherin to a receptor, or if the agent is an activity modulatingagent, reducing localization by, perhaps, less direct means. These arenot necessarily distinct groups as activity reducing agents mayencompass binding agents as well. However, as appreciated by one ofskill in the art, there are many methods in the art to help distinguishbetween direct binding reactions and perhaps a cascade of reactions thatis brought to a stop. A simple example would employ surface plasmonresonance, for example with a BiaCORE™ 2000 surface-plasmon-resonancedevice (BIAcore, Inc., Piscataway, N.J.) in order to observe if thepresence of the agent prevents binding by metadherin receptors; a sourceof said receptors would be lung cells identified in the current patent.Additionally, various reagents to stop well-known cascades as well asthe kinetics of the binding process, will all indicate which type ofagent one has discovered. However, it will often not be necessary todistinguish between these two agents.

In an alternative preferred embodiment, the method can be adjusted todetermine if possible expression-modulating agents can modulate theexpression of metadherin. In this embodiment, localization of cellstransformed with metadherin, with and without the possibleexpression-modulating agents are injected into a host and monitored forlocalization to the lungs. In this situation, it is important that theagents be given enough time to actually influence expression ofmetadherin, and that the agents are removed from the cell sample beforeinjection into the host to remove false positives that that merelysignify the presence of binding agents. If the expression-modulatingagents have an impact on the percent of cells that localize to the lungsfor the metadherin expressing cells, but not a significant effect on thelocalization of cells not expressing metadherin, then the agent will beclassified as an expression-modulating agent.

The small molecules, peptides, proteins, or antibodies derived from theabove screens, as well as the method of using them to prevent metastasisare also embodiments of this invention.

In another embodiment, cells expressing metadherin are used to screenfor their ability to bind to a library of cells, wherein the libraryexpresses DNAs that encode for potential metadherin receptor proteins.Creating a library of cells and analyzing the results can be performedin a variety of ways known to those of skill in the art. For example,once potential metadherin receptors are cloned, antibodies to thereceptors can be created in manner as described for metadherin. Theantibodies can then be used to assay which of the potential metadherinreceptors is an actual receptor.

Antibodies for use in the above methods may be prepared by any of avariety of techniques known to those of ordinary skill in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. In one such technique, an immunogen comprisingmetadherin is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep and goats). In this step, metadherinmay serve as the immunogen without modification. Alternatively, asuperior immune response may be elicited if the metadherin is joined toa carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. The immunogen is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the metadherin may then be purified from suchantisera by, for example, affinity chromatography using the metadherincoupled to a suitable solid support.

Monoclonal antibodies specific for metadherin may be prepared, forexample, using the technique of Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976, and improvements thereto. Briefly, these methodsinvolve the preparation of immortal cell lines capable of producingantibodies having the desired specificity (i.e., reactivity with themetadherin of interest). Such cell lines may be produced, for example,from spleen cells obtained from an animal immunized as described above.The spleen cells are then immortalized by, for example, fusion with amyeloma cell fusion partner, preferably one that is syngeneic with theimmunized animal. A variety of fusion techniques may be employed. Forexample, the spleen cells and myeloma cells may be combined with anonionic detergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against metadherin.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Monoclonal antibodies may also be used as therapeutic reagents, todiminish or eliminate lung or breast tumors. The antibodies may be usedon their own (for instance, to inhibit metastases) or coupled to one ormore therapeutic agents. Suitable agents in this regard includeradionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group that is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to oneantibody-molecule. In another embodiment, more than one type of agentmay be coupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers which provide multiple sites forattachment can be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of a resectedtumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibody used, thedensity of metadherin, and the rate of clearance of the antibody.

Diagnostic reagents of the present invention may also comprise DNAsequences encoding metadherin, or one or more portions thereof. Forexample, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify metadherinnucleic acids derived from a biological sample, wherein at least one ofthe oligonucleotide primers is specific for a metadherin gene or RNA.The presence of the amplified cDNA is then detected using techniqueswell known in the art, such as gel electrophoresis. Similarly,oligonucleotide probes specific for metadherin may be used in ahybridization assay to detect the presence of an inventive metadherin ina biological sample.

As used herein, the term “oligonucleotide primer specific formetadherin” means an oligonucleotide sequence that has at least about60%, preferably at least about 75% and more preferably at least about95%, identity to metadherin. Oligonucleotide primers and/or probes thatcan be usefully employed in the diagnostic methods described herein canhave at least about 10-40 nucleotides. The oligonucleotide primers cancomprise at least about 10 contiguous nucleotides of a polynucleotidehaving a sequence shown in SEQ ID NO: 2. Oligonucleotide probes for usein the diagnostic methods can have at least about 15 contiguousoligonucleotides of a polynucleotide with a sequence shown in SEQ ID NO:2. Techniques for both PCR based assays and hybridization assays arewell-known in the art. Primers or probes may thus be used to detectbreast tumor-specific sequences in biological samples, including blood,urine and/or breast tumor tissue.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of the cDNA of Metadherin

This example demonstrates how candidate cell adhesion proteins thatmediated breast cancer metastasis were identified using an in vivo phagescreening approach. This approach could also be used to find similarproteins or verify that possible metadherin variants are functional intheir ability to bind lung cells. The highly metastatic, BALB/c-derived4T1 mammary tumor cell line was selected to study tumor metastasesbecause 4T1 cells and human mammary adenocarcinomas share similar sitesof metastasis (Aslakson, C. J. and Miller, F. R., Cancer Res 52:1399-1405 (1992); Dexter, D. L. et al., Cancer Res 38: 3174-3181 (1978);Miller et al., 1983). Human breast cancer typically spreads first to thelungs in 24-77% of the cancers and to the liver in 22-62% (Kamby, C. etal., Cancer 59: 1524-1529 (1987); Rutgers, E. J. et al., Br J Surg 76:187-190 (1989); Tomin, R. and Donegan, W. L., J Clin Oncol 5: 62-67(1987)). Similarly, 4T1 cells spread in mice to the lungs in >95% ofcases and to the liver in >75% of the cancers (Pulaski, B. A. andOstrand-Rosenberg, S., Cancer Res 58: 1486-1493 (1998)). 4T1, a cellline derived from a BALB/c breast adenocarcinoma, was obtained from ATCCand maintained as described by Pulaski et al (Cancer Res 58: 1486-1493(1998)). MDA-MB-435 and KRIB cell lines were maintained as describedbefore (Laakkonen, P. et al., Nat Med 8: 751-755 (2002)). Nude BALB/cmice were subcutaneously injected with 1×10⁶ 4T1 tumor cells and keptfor 5 weeks (KRIB) or 10 weeks (MDA-MB-435). Tumors were then removed,frozen in OCT embedding medium (Tissue-Tek, Elkhart, Ind.), andsectioned. 4T1 cells were used to prepare a cDNA library enriched intranscripts that encode secreted and transmembrane proteins potentiallyinvolved in metastasis.

Phage Library and Screening

A cDNA library was prepared from membrane-bound polyribosomal mRNA of4T1 cells. Briefly, RNA from membrane-bound polysomes of 3.2×10⁸ 4T1cells was prepared using the methods described by Mechler (1987).Approximately 1 μg of this RNA was used to generate 6 μg of amplifiedanti-sense mRNA (aRNA), using the methods described by Luo et al.(1999). Using aRNA as template, mRNA was synthesized as described by Luoet al. (1999), except the primer, 5′-TTNNNNNN-3′ [SEQ ID NO: 4], wasused instead of random hexamer primer, and methylated dNTPs were usedinstead of dNTPs. A cDNA library was prepared from the mRNA, asdescribed in the manufacturer's protocol (OrientExpress™ Random PrimercDNA synthesis kit; Novagen, Madison, Wis.).

A “stop linker” was inserted into the myc epitope phage vectorsdescribed above to prevent myc epitope expression in phage vectors thatwere unsuccessfully ligated to cDNA during library construction.Oligonucleotides, encoding stop codons in all three reading frames, weresynthesized, phosphorylated, annealed, and ligated toEcoRI/HindIII-digested myc epitope phage vectors to form myc-T7 vectors.The cDNA libraries were then ligated to EcoRI/HindIII-digested myc-T7vectors, phage were packaged, and libraries were amplified. As measuredby plaque assay, the library contained 4.7×10⁶ primary recombinants.

T7 phage vectors that expressed myc epitope-tagged inserts of the cDNAin the library were then assembled. Oligonucleotides, encoding mycepitopes in all three reading frames, internal EcoRI and HindIIIrestriction enzyme cleavage sites, and flanking EcoRI/HindIII adapterswere synthesized. The oligonucleotides were then individuallyphosphorylated, annealed, and ligated to EcoRI/HindIII-digested T7Select1-2a, 1-2b, or 1-2c vector arms (Novagen) to generate myc epitope phagevectors.

Phage clones that expressed cDNA inserts with open reading frames wereenriched by three rounds of selection with an anti-myc mAb (3.1 μg/mlMAB8864; Chemicon, Temecula, Calif.) bound to rat anti-mouse IgG1magnetic beads (3.1 μl per 1 ml of buffer; Miltenyi Biotec, Auburn,Calif.). Selections were performed with 10¹¹ pfu of phage in 10 ml ofDulbecco's phosphate buffered saline containing 0.5% bovine serumalbumin (PBSB). Phage were applied to a magnetized LS MACS column(Miltenyi Biotec), washed with buffer (50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate),eluted with PBSB after demagnetizing the column, and transferred to asecond column for more washes. Phage were amplified using the liquidlysate method after each anti-myc selection round.

Ex vivo and in vivo screenings with the 4T1 cDNA library were performedas previously described (Hoffman et al., in press). The library waspre-selected for phage that bound to lung tissue by performing tworounds of ex vivo selection on single-cell lung suspensions. Thepre-selected 4T1 library was then injected intravenously into Balb/cmice and phage that accumulated in lung were isolated. After threerounds of in vivo selections, cDNA inserts were sequenced from 32 phageclones.

Individual phage clones were tested for their ability to specificallybind to lung vasculature. One of the five lung-specific clonesidentified encoded a fragment of a protein recently deposited intoGenBank™ (accession numbers AAL92861 and AAP30791). The selected phage,when intravenously (i.v.) injected into mice and allowed to circulatefor five minutes, bound to lungs almost 20-fold more than control phage(FIG. 3, error bars represent mean±s.d. for 2-7 experiments pervariable). No selective accumulation of this phage was seen in breast,skin, brain, or liver and its amount was less than 2-fold above controlphage in pancreas and kidney. The phage co-localized with blood vesselsin the lungs. This was determined via an anti-phage immunostaining oflungs from mice co-injected with fluorescein-labeled tomato lectin andeither metadherin phage (T7-metadherin) or T7-415 non-recombinant phage(T7-Control). Control lungs were from non-injected mice (lectin⁻phage⁻),mice injected with lectin alone (lectin⁺phage⁻), and mice only injectedwith metadherin phage (lectin⁻phage⁺). Anti-phage antibody was detectedwith Alexa 594 goat anti-rabbit IgG antibody. Nuclei were stained withDAPI. This demonstrates that the protein fragment was an effective meansfor delivering desired substances to blood vessels in the lungs.

Cloning of Full-Length Metadherin cDNA

The following primers were synthesized to amplify the full-length mousemetadherin cDNA:

(SEQ ID NO: 5) 5′-ACCATGGCTGCACGAAGCTGGCAGGACGAGCTG-3′. (SEQ ID NO: 6)5′-TCACGTTTCCCGTCTGGCCTTTTTCTTCTTTTTTA-3′.

RNA was isolated from 4T1 cells using a Total RNA isolation kit (Qiagen,Valencia, Calif.). The metadherin cDNA (SEQ ID NO: 2) was amplified byRT-PCR using a Superscript™ One-Step RT-PCR Kit for Long Templates(according to manufacturer's protocol; Invitrogen, Carlsbad, Calif.) andsubcloned into the TOPO-TA vector, pcDNA3.1-V5/His according to themanufacturer's protocol (Invitrogen, Carlsbad, Calif.).

Example 2 Antibodies Against Metadherin

This example demonstrates how to obtain antibodies directed againstmetadherin, in particular, the lung-homing domain of metadherin. Thisexample also demonstrates that metadherin is localized on the externalsurface of tumor cells. Anti-T7 phage affinity purified antibody waspreviously described (Laakkonen, et al., (2002). Nat Med 8: 751-755). Apolyclonal antibody was generated in New Zealand White rabbits againstthe recombinant metadherin lung-homing domain (SEQ ID NO: 3) that wasfused to glutathione-S transferase. The initial immunization was done incomplete Freund's adjuvant and boosters were with incomplete Freund'sadjuvant. The antibody was affinity purified on recombinanthexahistine-tagged metadherin₍₃₇₈₋₄₄₀₎ peptide coupled to SulfoLink Gel(Pierce, Rockford, Ill.) via a cysteine residue added to the aminoterminus of the metadherin₍₃₇₈₋₄₄₀₎ peptide. The tags were added byfirst subcloning the lung-binding domain into the vector pQE-60 (Qiagen,Valencia, Calif.) using the primers5′-CCCGCCATGGGGTTAAATGGTTTGTCTTCTGCTGACCC-3′, (SEQ ID NO: 7); and5′-CCCGAGATCTTTTAGATTTCCCAGTTGGAAGAGCTCCCTCCCC-3′ (SEQ ID NO: 8).

These primers resulted in 6 histidine residues at the C-terminus whenexpressed in the pQE-60 vector. This construct was then used forsubcloning, via PCR, the lung-binding domain with hexahistidine tag intothe PGEX4T1 vector (Pharmacia, Piscataway, N.J.), using primers5′-CCCGGGATCCGGGTGCGGGTTAAATGGTTTGTCTTC-3′ (SEQ ID NO: 9) and5′-CCCGCTCGAGTTAGTGATGGTGATGGTGATGAGATCTTTTAG-3′ (SEQ ID NO: 10). Thisresulted in a GST tag at the N-terminus.

These antibodies bound to non-permeabilized 4T1 cells in flow cytometry(FIG. 4A, anti-myc antibodies were applied to the cells and detectedwith a PE-labeled secondary Ab). This result confirms the presence ofthe lung-homing domain of metadherin on tumor cells at the cell surfacewhere it would be available to bind to vascular targets duringmetastasis. A control antibody against a cytoplasmic protein (Bcl-2) andrabbit IgG did not bind to the surface of non-permeabilized 4T1 cells,while the cells were strongly positive for integrin α5β1 (FIG. 4A,control cells were stained with FITC-labeled secondary Ab and propidiumiodide alone (FITC/PI only)).

Example 3 FACS Detection of Metadherin Via Metadherin Antibodies

This example demonstrates how to detect the presence of metadherinprotein on cells. To analyze the presence of metadherin on 4T1 cells byFACS, the cells were detached from culture plates by incubating with PBSwith 2 mM EDTA (PBSE) for 10 minutes. The cells were then washed withPBSB and incubated with 40 μg/ml (in PBSB) of the following antibodies:anti-Bcl2 (SL-492, Santa Cruz Biotechnology, Santa Cruz, Calif.), normalrabbit IgG (Sigma, St. Louis, Mo.), anti-integrin α₅β₁ (ProteinG-purified from rabbit serum containing antibodies raised against humanfibronectin receptor), and anti-metadherin₍₃₇₈₋₄₄₀₎. To detect boundantibodies, cells were incubated with goat anti-rabbit IgG-FITC (40μg/ml in PBSB; Molecular Probes, Eugene, Oreg.). After the final wash,the cells were resuspended with PBS containing 2 μg/ml of propidiumiodide (PI) and analyzed by FACS. The antibodies raised againstmetadherin₍₃₇₈₋₄₄₀₎ specifically bound to non-permeabilized 4T1 cells inflow cytometry (FIG. 4A). In contrast, the control antibodies against acytoplasmic protein (Bcl-2) and rabbit IgG did not bind to the surfaceof non-permeabilized 4T1 cells.

Example 4 FACS analysis of Metadherin Via MYC-Metadherin

This example demonstrates an alternative method by which a variant ofmetadherin can be observed by FACS analysis. A fusion protein,myc-metadherin, which was still able to localize metadherin to the lungswas used in the following experiment. Transiently transfected HEK293Tcells expressing myc-vimentin, myc-metadherin, or myc-pCMV vector alone(Clontech, Palo Alto, Calif.) were detached from their culture dishes bygently washing with PBS containing 1% BSA (PBSB). The cells were thenstained with mouse anti-myc mAb (2 μg/ml in PBSB; Chemicon, Temecula,Calif.) for 20 minutes at 4° C. The cells were washed with PBSB, stainedwith goat anti-mouse IgG PE-labeled antibody (4 μg/ml in PBSB;Pharmingen, San Diego, Calif.), washed again with PBSB, fixed with 2%paraformaldehyde in PBS, resuspended in PBS, and analyzed using aFACScan flow cytometer (BD, San Jose, Calif.). It was observed thatintact myc metadherin-expressing cells were specifically labeled withanti-myc antibodies (FIG. 4B). However, the cells treated withmyc-vimentin or myc-pCMV were not labeled with the anti-myc antibody.

Example 5 Blood Vessel Localization via Metadherin

This example demonstrates how to examine for the presence of metadherinin lung blood vessels and that a metadherin expressing phage will causecell delivery to the lung blood vessels. Phage expressing metadherinwere examined by i.v. injection of 2.5×10¹⁰ pfu metadherin phage (in 200μM9LB) into the tail vein of a mouse. Blood vessels were visualized byco-injection of phage with 200 μg of Lycopersicon esculentum (tomato)lectin conjugated to fluorescein and either metadherin phage(T7-metadherin) or T7-415 non-recombinant phage (T7-Control). Controllungs were from non-injected mice (lectin⁻phage⁻), mice injected withlectin alone (lectin⁺phage⁻), and mice only injected with metadherinphage (lectin⁻phage⁺). Anti-phage antibody was detected with Alexa 594goat anti-rabbit IgG antibody. Nuclei were stained with DAPI. Theinjected materials were allowed to circulate for 10 min. Lungs wereremoved and frozen in OCT embedding medium (Tissue-Tek). The phage boundto lungs almost 20-fold more than control phage (FIG. 3). No selectiveaccumulation of this phage was seen in breast, skin, brain, or liver andits amount was less than 2-fold above control phage in pancreas andkidney. The phage co-localized with blood vessels in the lungs.

Example 6 Analysis of Metadherin and Metadherin Variant Levels in TumorCell Lysates

This example demonstrates how to monitor the amount of metadherin intumor cell lysates. Tumor cell lysates were prepared in 2.5× Laemmli'ssample buffer (Laemmli, 1970) at a ratio of 10⁶ cells per 150 μl,subjected to SDS/PAGE on 4-20% acrylamidé gradient gels. Proteins weretransferred to PVDF membrane and immunoblots were performed withanti-metadherin₍₃₇₈₋₄₄₀₎ (0.1 μg/ml) and goat anti-rabbit IgG-HRP(diluted 1:10,000; Bio-Rad, Hercules, Calif.) and developed usingECL+plus chemiluminescence reagent (Amersham Biosciences, Piscataway,N.J.), according to the manufacturer's instructions. The relative amountof metadherin detected by immunoblot was quantitated using anAlphaImager (Alpha Innotech, San Leandro, Calif.). β-actin was detectedwith an anti-actin monoclonal antibody (10 μg/ml, Chemicon, Temecula,Calif.). Transferrin receptor was detected with an anti-transferrinreceptor polyclonal antibody (2 μg/ml, Santa Cruz Biotech, Santa Cruz,Calif.). Affinity-purified polyclonal antibody reactive to a novel 175kDa protein Clone D2 was prepared as described foranti-metadherin₍₃₇₈₋₄₄₀₎. Control immunoblots were performed withanti-Clone D2 (0.1 μg/ml) and goat anti-rabbit IgG-HRP (describedabove).

In 4T1 tumor cell extracts, anti-metadherin₍₃₇₈₋₄₄₀₎ antibodies detectedproteins with apparent molecular weights of approximately 80 kDa, 75kDa, and 55 kDa. KRIB and MDA-MB-435 cell extracts also contained the 80kDa and 75 kDa proteins. FIG. 5 shows an immunoblot of endogenousmetadherin. Lanes 1 and 4, are from 4T1 cell extract, lane 2, KRIB cellextract, and lane 3, MDA-MB-435 cell extract. Immunoblot detection ofmetadherin was performed with anti-metadherin₍₃₇₈₋₄₄₀₎ (lanes 1-3).Immunoblot detection of an unrelated protein, Clone D2, was performedwith anti-Clone D2 polyclonal antibody (lane 4). The controlaffinity-purified polyclonal antibody reactive to a non-related protein(Clone D2) did not detect the anti-metadherin₍₃₇₈₋₄₄₀₎ immunoreactivebands. The 80 kDa and 55 kDa proteins detected byanti-metadherin₍₃₇₈₋₄₄₀₎ were also produced by an in vitro transcriptionand translation reaction using an epitope-tagged metadherin cDNA astemplate; this suggested that the 75 kDa and 55 kDa proteins may bedegradation products of metadherin.

Example 7 Metadherin Localizes to the Cell Membrane and the Specificityof mAB₍₃₇₈₋₄₄₀₎

This example provides a demonstration of how to useanti-metadherin₍₃₇₈₋₄₄₀₎ antibodies to look for metadherin in differentlocations of the cell, and describes the distribution of the moleculethroughout the cell. This example also demonstrates how to determinewhich labeling effects are due to the specific binding of this antibody.

Cell-surface labeling: Anti-metadherin₍₃₇₈₋₄₄₀₎, diluted to 20 μg/ml inice-cold IMEM (Invitrogen) with 10% fetal bovine serum (FBS), was addedto cells cultured on chamber slides and incubated for 1 hour on ice. Thecells were washed with IMEM and fixed with cold 4% paraformaldehyde inPBS for 15 minutes. Anti-metadherin antibodies were detected with Alexa594 goat anti-rabbit IgG (diluted 1:500 in PBS with 1% FBS and 3% goatserum). Slides were mounted with Vectashield fluorescence mountingmedium (Vector, Burlingame, Calif.). In non-permeabilized cells, thestaining was concentrated at the edges of the cells. Controls showedthat pre-incubation of anti-metadherin₍₃₇₈₋₄₄₀₎ with themetadherin₍₃₇₈₋₄₄₀₎ lung-homing peptide inhibited the staining, whereasa control peptide did not.

Permeabilized cell labeling: Cells were first fixed with 4%paraformaldehyde (described above) and then treated with 0.1% TritonX-100 in PBSB for 15 minutes. The cells were washed with PBSB andincubated with anti-metadherin₍₃₇₈₋₄₄₀₎ (diluted to 20 μg/ml in IMEMwith 10% FBS) for 1 hour at room temp. Anti-metadherin₍₃₇₈₋₄₄₀₎ wasdetected with Alexa 594 goat anti-rabbit IgG, as described above. Infixed and permeabilized 4T1 cells, metadherin immunoreactivity localizedto the cytoplasm and plasma membrane. In sections (0.15 μm thick) ofnon-permeabilized 4T1 cells stained with anti-metadherin₍₃₇₈₋₄₄₀₎ thestaining was concentrated to the edges of the cell. As a control,non-permeabilized 4T1 cells that were pre-incubated with excessmetadherin₍₃₇₈₋₄₄₀₎ peptide demonstrated reduced staining withanti-metadherin₍₃₇₈₋₄₄₀₎, while those cells incubated in excess controlpeptide did not. Alexa 594 goat anti-rabbit IgG antibody were used.

Example 8 Detection of Metadherin in Human Tumors Samples as a Means forDiagnosing the Spread of Cancer

This example demonstrates how to determine the amount of metadherinpresent in a human tissue sample that is cancerous. Paraffin-embeddedhuman tumor sections (Spring Biosciences, Fremont, Calif.) werede-paraffinized and then treated with Target Retrieval Solution(according to the manufacturer's instructions; Dako, Carpinteria,Calif.). The sections were stained as described above, except PBSB wassubstituted for 0.5% Blocking Reagent (NEN Life Sciences, Boston, Mass.)in 0.1M Tris/150 mM NaCl. To determine specificity,anti-metadherin₍₃₇₈₋₄₄₀₎ (20 μg/ml) was pre-incubated overnight with 200μg/ml recombinant metadherin lung-homing protein (SEQ ID NO: 3) orunrelated recombinant control protein (72 amino acid, lung-homing CloneD2) in blocking buffer before immunostaining the sections. Both sectionsof human breast tumor and normal human breast tissue were stained witheither anti-metadherin₍₃₇₈₋₄₄₀₎ alone or anti-metadherin₍₃₇₈₋₄₄₀₎pre-incubated with excess metadherin₍₃₇₈₋₄₄₀₎ peptide or excess controlpeptide.

Several human breast cancer sections stained withanti-metadherin₍₃₇₈₋₄₄₀₎ showed high expression of metadherin throughoutthe tumor. In contrast, no cytoplasmic or cell surface-associatedmetadherin was detected in normal human breast tissue; however, nuclearstaining was present. The cell surface staining of breast cancer tissuewas inhibited with the metadherin₍₃₇₈₋₄₄₀₎ peptide, but not with acontrol peptide. Neither peptide inhibited nuclear staining.

Example 9 Detection of Metadherin in Xenografts as Means for DiagnosingCancer Cells

This example demonstrates that the detection method works for samplesthat are not of human origin. High amounts of metadherin proteins weredetected in sections of murine MDA-MB-435 (breast adenocarcinoma) andKRIB (osteosarcoma tumor) tumor xenografts, which are two tumor modelsknown to generate lung metastases (Berlin et al., 1993; Price et al.,1990). Xenografts were grown in nude Balb/c mice and were analyzed byimmunostaining. Sections were stained with eitheranti-metadherin₍₃₇₈₋₄₄₀₎ alone or anti-metadherin₍₃₇₈₋₄₄₀₎ pre-incubatedwith excess metadherin₍₃₇₈₋₄₄₀₎ peptide or excess control peptide.Anti-metadherin₍₃₇₈₋₄₄₀₎ was detected with Alexa 594 goat anti-rabbitIgG antibody and nuclei were stained with DAPI. Metadherin appeared tobe localized to the tumor cell surface in the both models andparticularly strong expression of metadherin was found at the peripheryof the KRIB tumors. The anti-metadherin immunostaining was specific,since pre-incubation of antibody with the metadherin₍₃₇₈₋₄₄₀₎lung-homing peptide, but not the control peptide, inhibited thestaining. Subcutaneous tissue or skin adjacent to the tumors showed noanti-metadherin staining.

Specific metadherin staining was present in frozen tissue sections ofnormal mouse mammary tissue at the apical surface of epithelial cellslining ducts of the mammary glands and a small amount of metadherin wasdispersed through the mammary fat pad. Sections of mouse breast tissuewere stained with either anti-metadherin₍₃₇₈₋₄₄₀₎ alone oranti-metadherin₍₃₇₈₋₄₄₀₎ pre-incubated with excess metadherin₍₃₇₈₋₄₄₀₎peptide. No metadherin was detected in the spleen, kidney, lung, andskin, but minute amounts were seen in the liver. Purkinje neurons in theearly post natal and adult cerebellum were strongly positive formetadherin staining. These immunostaining results show that metadherinis selectively over-expressed in tumors.

Example 10 Delivery of a Desired Substance to a Particular Location

This example demonstrates the ability of metadherin to effectivelydeliver an associated substance to the lung tissue. To test the effectof metadherin on tissue distribution of i.v.-injected tumor cells,HEK293T cells were studied by transiently transfecting them withmetadherin cDNA (SEQ ID NO: 2). HEK293T cells were co-transfected withDsRed2 (Clontech, Palo Alto, Calif.) and either metadherin-pCMV or anempty myc-pCMV vector. Two days post-transfection, the cells weredetached with PBSE and filtered through a 40 μm nylon filter.DsRed-expressing cells were isolated using a FACS Vantage flow cytometer(BD Biosciences, San Jose, Calif.). 2.5×10⁵ DsRed-positive cells, thecells being the desired substance in this experiment, were injected intothe tail-vein of nude BALB/c mice. Five mice were injected with eachcell type. After 2 hours, the mice were sacrificed, organs were removedand fixed with 4% paraformaldehyde in PBS, and 10 μm-thick frozen tissuesections were prepared. For each lung section, 3 different fields werecounted. Clumps of DsRed positive cells with 3 or more cells wereexcluded from the count. Five sections per lung were counted.

Fluorescent cells were detected in the blood vessels of lungs examinedtwo hours after the injection; cell counting showed 22% moremetadherin-transfected cells than that of vector-transfected cells. Nosignificant amounts of DsRed2 HEK293T cells were observed in the brain,skin, liver, kidney, heart, spleen, or pancreas. FIG. 6 shows the numberof DsRed2-positive cells per viewing field in the lung sections (n=75;One-tailed Student's t-test; *P<0.001). This result supports the phagehoming data indicating that metadherin preferentially binds to lungvasculature.

Example 111 Inhibition of Metadherin via SiRNA and its Ability to Reducethe Formation of Metastases siRNA to metadherin can inhibit metadherin

The ability of siRNA to alter metadherin production was examined. Asshown in FIG. 7A, siRNA reactive to metadherin, but not to GAPDH or anegative control mRNA (e.g., “scrambled”), was able to reduce expressionof transfected myc-metadherin in HEK293T cells. Metadherin levelsreturned to normal after two weeks. However, a workable model wascreated using the co-expression of EGFP and the metadherin-reactivesiRNA or scrambled-siRNA in 4T1 cells and the cells were selected byFACS. The transfection with metadherin-siRNA did not affect theexpression of beta-actin or the type-II transmembrane protein,transferring receptor (see FIG. 7B). However, metadherin proteinexpression in metadherin-siRNA cells was reduced by about 40%, relativeto the scrambled-siRNA cells (FIG. 7C). The arrow in FIG. 7C denotes the80 kDa metadherin protein band quantified by densitometry.

Measured by real-time PCR, metadherin-siRNA cells expressed about 40%less metadherin mRNA than the scrambled-siRNA cells when metadherin mRNAlevels were normalized to beta-actin mRNA levels (FIG. 7D). The relativeamount of metadherin mRNA was normalized to the abundance of β-actinmRNA, also detected by real time RT-PCR. Error bars represent mean±s.d.

siRNA to Metadherin can Inhibit the Formation of Metastases in MouseModel

This example demonstrates the effectiveness of siRNA directed againstmetadherin to alter the metastatic potential of breast cancer cells byreducing the native level of metadherin in cells. For the siRNA-mediatedinhibition of metadherin expression, nucleotides 1597-1615 of the mousemetadherin cDNA (5′-GTGCCACCGATGTTACAAG-3′) (SEQ ID NO: 11) were used asthe target sequence. Oligonucleotides containing this target sequencewere synthesized and subcloned into the pSilencer 3.0-H1 plasmid(Ambion, Austin, Tex.), according to the manufacturer's instructions.Green fluorescent protein (EGFP) was co-expressed with themetadherin-reactive siRNA or a control scrambled-siRNA in 4T1 cells andselected for siRNA-transfected cells by FACS. 4T1 cells were transfectedwith the metadherin or a negative control siRNA pSilencer vectortogether with an EGFP-expression vector (Clontech, Palo Alto, Calif.),using a 4:1 ratio of pSilencer to EGFP vectors. Two dayspost-transfection, 4T1 cells that were labeled with EGFP and excludedpropidium iodide were isolated by FACS. 10,000 or 50,000 of theseselected 4T1 cells in 100 μl of PBS were injected into the tail-vein ofanesthetized nude BALB/c mice. The mouse lungs were harvested 22 dayspost-injection and stained with Bouin's solution. The tumor foci on thelung surface were counted under a dissecting microscope. Data wererecorded as the number of tumor foci formed per 10,000 cells injected.

When injected into mice, the 4T1 cells expressing metadherin-reactivesiRNA formed about 80% fewer experimental lung metastases than cellsexpressing scrambled-siRNA (P<0.001). The results are displayed in FIG.7E. Values are expressed as number of tumor foci per 10,000 cellsinjected. Bars represent mean±s.e.m (One-tailed Student's t-test;*P<0.02,**P<0.01, ***P<0.001). Percentages indicate relative suppressioncompared to control group. No difference in the viability ofmetadherin-siRNA and scrambled-siRNA cells was detected by the use ofpropidium iodide staining. In addition, a count of the number of cellsbefore and two days after transfecting the siRNA expression plasmids didnot show any significant effect of the metadherin-siRNA on the growthrate of the cells.

Example 12 Production of an Additional Homolog to Metadherin

The deduced amino acid sequence of the lung-homing domain of thelung-homing phage is shown in SEQ ID NO: 3. Using BLAST (Altschul, S.F., et al., Nucleic Acids Res 25: 3389-3402 (1997)), It was determinedthat one cDNA clone (GenBank™ accession number AY082966—SEQ ID NO: 16)encoded the putative full-length human protein (SEQ ID NO: 13)corresponding to the phage clone. The cDNA encoding metadherin has acoding region that is 93% identical to the coding region (SEQ ID NO: 2)of the murine metadherin. The GenBank™ entry refers to the protein as“LYRIC” and describes it as a putative CEACAM1-associated protein incolon carcinoma. These results that show the importance of thislung-homing protein in breast cancer metastasis. The protein was namedmetadherin (metastasis adhesion protein). A reported mouse cDNA homologof metadherin was used (GenBank™ accession number AK029915) to designoligonucleotides and amplified by reverse transcription-polymerase chainreaction the full-length mouse metadherin cDNA (SEQ ID NO: 2). As shownin SEQ ID NO: 2, the mouse metadherin cDNA is 2530 nucleotides in lengthwith a 1737 base pair coding region that starts at nucleotide 319 andends at nucleotide 2058. The human metadherin cDNA is 2031 nucleotidesin length with a 1748 base pair coding region that starts at nucleotide79 and ends at nucleotide 1827. As will be appreciated by one of skillin the art, this procedure can readily be repeated to identifyadditional homologs in other organisms.

Example 13 Process for Determining the Functional Regions of Metadherin

This example demonstrates a method for identifying and then verifyingthe functional features of metadherin or its variants. Analysis of thehydrophobic regions of metadherin (Kyte, J. and Doolittle, R. F., J MolBiol 157, 105-132 (1982)) revealed that amino acid residues 52-74 encodea putative transmembrane domain (e.g., FIG. 1B and FIG. 1C). A searchdid not reveal any domains in metadherin that were similar to otherknown proteins. Using a hidden Markov model to detect membrane helicesand predict transmembrane topology in proteins (Glasgow, J. (1998),Proceedings, Sixth International Conference on Intelligent Systems forMolecular Biology: June 28-Jul. 1, 1998, Montreal, Quebec (Menlo Park,Calif., AAAI Press); Krogh, A., et al., J Mol Biol 305: 567-580 (2001)),it was discovered that metadherin was predicted to be a type IItransmembrane protein with an extracellular lung-homing domain. Toconfirm this prediction, a c-myc epitope was subcloned into thelung-homing domain of the metadherin cDNA, as shown in FIG. 1C. A mycepitope was added to metadherin protein by first inserting an EcoRIrestriction enzyme site in the metadherin cDNA after nucleotide 1222with a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla,Calif.). Then, oligonucleotides encoding a myc epitope (EQKLISEEDL) [SEQID NO: 12] and flanking EcoRI adapters were synthesized, phosphorylated,and ligated into the EcoRI-digested metadherin cDNA. The myc-metadherincDNA was subcloned into the pCMV vector (Clontech, Palo Alto, Calif.).Human myc-vimentin cDNA was generated by reversetranscription-polymerase chain reaction, using vimentin-specific primersand human mRNA as template, and then subcloned into the pCMV-Myc vector(Clontech, Palo Alto, Calif.).

This myc-tagged cDNA was expressed in HEK293T cells and these cells werethen stained with anti-myc antibodies. Using flow cytometry, it wasobserved that intact myc metadherin-expressing cells were labeled withanti-myc antibodies, which indicated that the lung-homing domain ofmetadherin was extracellular. No cell-surface labeling was detected invector-transfected cells or non-permeabilized cells expressing theintracellular protein, myc-vimentin. Anti-myc antibodies stained the mycvimentin-expressing cells when permeabilized, confirming the expressionof myc-vimentin, and permeabilized cells expressing vector alone werenot stained with anti-myc antibodies.

This example can be modified by simply using the antibody tometadherin₍₃₇₈₋₄₄₀₎ in order to determine if variants of metadherin arestill functional or still have certain desired properties.

Example 14 Inhibition of Metastasis by Metadherin mAbs

This example demonstrates how metastasis were inhibited by the additionof a binding agent, in this case, antibodies directed to thelung-binding domain of metadherin. The mouse model system was used toinhibit metadherin activity in the 4T1 cells with antibodies reactive tothe lung-homing domain of metadherin. The 4T1 cells were detached fromplates with PBSE, washed once with PBS, re-suspended to 5×10⁵ cells/mlin PBS, and placed on ice. Anti-metadherin₍₃₇₈₋₄₄₀₎ or rabbit IgG (200μg) was added to 5×10⁴ cells and the cells were then injected via thelateral tail vein into female Balb/c nu/nu mice. Animals were sacrificed7 days after tumor cell injection. Lungs were recovered, stained withBouin's solution, and the tumor foci on the surface of the left lobewere counted under a dissecting microscope. When co-injected with the4T1 cells, anti-metadherin₍₃₇₈₋₄₄₀₎ inhibited lung metastasis by about40%, compared to 4T1 cells treated with rabbit IgG (FIG. 7F). In aseparate experiment, no difference was observed between the growth ofmammary fat pad tumors formed from 4T1 cells pretreated with theanti-metadherin₍₃₇₈₋₄₄₀₎ or rabbit IgG.

Example 15 Screening of Therapeutics that Block Metastasis viaMetadherin

This example demonstrates how one can screen for small molecules,peptides or proteins that bind to and block metastasis via blockingmetadherin. Cells expressing metadherin or a fragment thereof, such asin the phage clones in Example 1, are mixed with the possible blockingagent and then injected into mice, according to Example 1. If theinjected cells localize to the lung tissue, then the blocking agent isnot effective as a complete blocking agent, over the time period of theexperiment. A reduction in the localization of the cells to the lungtissue or comparison to a control will indicate a possible blockingagent.

Example 16 Determination of Metadherin's Role in Other Cancers, inSilico

This example demonstrates how one can determine if the metadherin of thecurrent invention will be useful in stopping the metastasis of othercancers. SAGEmap (Lal, A., et al., Cancer Res 59: 5403-5407 (1999);Lash, A. E., et al., Genome Res 10: 1051-1060 (2000)), a component ofThe Cancer Genome Anatomy Project at the National Center forBiotechnology Information, was used to determine that metadherin wassignificantly over-expressed not only in breast cancers, but also incancers of the brain and prostate (P<0.05). This suggests a similar rolefor metadherin in the metastasis of brain and prostate cancers. One maythen administer the anti-metadherin antibody of the current inventionand determine if cancer metastasis is halted or not.

Example 17 Determination of Variants of Metadherin, in Silico

This example demonstrates a technique to determine other variants ofmetadherin. A BLAST algorithm (Altschul, S. F., et al., Nucleic AcidsRes 25: 3389-3402 (1997)), was used to determine additional mouse andhuman metadherin-like molecules in the GENBANK™ databases. FIG. 2demonstrates a functional comparison of the several metadherin homologsor variants from human (SEQ ID NO: 13), mouse (SEQ ID NO: 1), rat (SEQID NO: 14), and zebra fish (SEQ ID NO: 15) are now known.

Example 18 Determination of Functional Variants for PreventingMetastasis

This example demonstrates how possible variants of metadherin can beexamined to determine if they are still functional for preventing cancermetastasis. The possible variant is expressed in a host cell that can bemonitored, as in Example 1. The variant is then administered, as inExample 1, into a host to determine if the variant is able to localizethe host cell to the lung tissue. If the variant is still able tolocalize to the lung tissue, and thus bind to the metadherin receptors,then it is considered a functional variant, for the purposes ofpreventing the spread of cancer.

Example 19 The Use of Antibodies Directed to Metadherin to Reduce BreastCancer Metastasis

A patient with cancer and a risk of breast cancer metastasis isselected. The risk of breast cancer metastasis is examined bydetermining the presence of metadherin in the patient's system, withincreased levels of metadherin, relative to cancer free patients,indicating that the patient has an increased risk of breast cancermetastasis. An effective amount of an antibody directed to the bindingregion of metadherin is then administered to the patient, this may beadministered in combination with other cancer chemotherapy or othertreatments. An effective amount is determined on a patient by patientbasis and can be directly monitored by repeated assays for antibody-freemetadherin that still exists in the patient's system. Alternatively, aneffective amount is determined by monitoring the overall health of thepatient and the reduction of any spread of the cancer. The treatment iscontinued until the spread of the cancer is reduced.

Example 20 The Use of siRNA Directed to Metadherin to Reduce BreastCancer Metastasis

A patient with cancer and a risk of breast cancer metastasis isselected. The risk of breast cancer metastasis is determined by lookingfor the presence of metadherin in the patient's system, with increasedlevels of metadherin, relative to cancer free patients, indicating thatthe patient has an increased risk of breast cancer metastasis. Aneffective amount of a siRNA directed to metadherin is then administeredto the patient, this may be administered in combination with othercancer chemotherapy or other treatments. An effective amount isdetermined on a patient by patient basis and is directly monitored byrepeated assays for the presence of metadherin that still exists in thepatient's system. The treatment is continued until the spread of thecancer is reduced.

Example 21 The Use of siRNA to Treat Breast Cancer

A patient with breast cancer is selected. The risk of breast cancermetastasis is determine by looking for the presence of metadherin in thepatient's system, with increased levels of metadherin, relative tocancer free patients, indicating that the patient has an increased riskof breast cancer metastasis. An effective amount of a siRNA directed tometadherin is then administered to the patient, this may be administeredin combination with other cancer chemotherapy or other treatments. Aneffective amount is determined on a patient by patient basis and isdirectly monitored by repeated assays for the presence of metadherinthat still exists in the patient's system. The treatment is continueduntil the breast cancer is reduced.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference in their entireties.

1-24. (canceled)
 25. A method of delivering an agent to the lungvasculature of a patient, comprising: linking an agent to a metadherinprotein to create a metadherin targeted agent; and administering saidmetadherin targeted agent to a patient, thereby delivering an agent to alung vasculature of the patient.
 26. The method of claim 25 wherein saidmetadherin has an amino acid sequence shown in SEQ ID NO:
 17. 27. Themethod of claim 26, wherein said lung-binding domain has an amino acidsequence that is at least 95% identical to the amino acid sequence shownin SEQ ID NO:
 17. 28. The method of claim 25, wherein administering saidmetadherin targeted agent comprises intravenous administration of saidtargeted composition. 29-44. (canceled)
 45. The method of claim 25,wherein said lung-binding domain has an amino acid sequence that is atleast 99% identical to the amino acid sequence shown in SEQ ID NO: 17.46. The method of claim 25, wherein metadherin comprises the amino acidsequence of SEQ ID NO:
 13. 47. A method of delivering an agent to thelung vasculature of a patient, said method comprising administering anagent linked to a metadherin protein to a patient, thereby delivering anagent to a lung vasculature of the patient.
 48. The method of claim 47,wherein the metadherin protein comprises the amino acid sequence of SEQID NO:
 13. 49. The method of claim 47, wherein the metadherin proteincomprises the amino acid sequence of SEQ ID NO:
 17. 50. The method ofclaim 49, wherein said lung-binding domain has an amino acid sequencethat is at least 95% identical to the amino acid sequence shown in SEQID NO:
 17. 51. The method of claim 49, wherein said lung-binding domainhas an amino acid sequence that is at least 99% identical to the aminoacid sequence shown in SEQ ID NO:
 17. 52. The method of claim 47,wherein the agent is a therapeutic agent.
 53. The method of claim 47,wherein the agent is a cancer chemotherapeutic agent.
 54. The method ofclaim 47, wherein the agent is a cancer chemotherapeutic agent fortreatment of breast cancer.
 55. The method of claim 47, wherein theagent is a diagnostic agent.
 56. The method of claim 47, wherein theagent is a detectable agent.